Array cytometry

ABSTRACT

A method and apparatus for the manipulation of colloidal particulates and biomolecules at the interface between an insulating electrode such as silicon oxide and an electrolyte solution. Light-controlled electrokinetic assembly of particles near surfaces relies on the combination of three functional elements: the AC electric field-induced assembly of planar aggregates; the patterning of the electrolyte/silicon oxide/silicon interface to exert spatial control over the assembly process; and the real-time control of the assembly process via external illumination. The present invention provides a set of fundamental operations enabling interactive control over the creation and placement of planar arrays of several types of particles and biomolecules and the manipulation of array shape and size. The present invention enables sample preparation and handling for diagnostic assays and biochemical analysis in an array format, and the functional integration of these operations. In addition, the present invention provides a procedure for the creation of material surfaces with desired properties and for the fabrication of surface-mounted optical components. This invention is also for a method and apparatus to direct the lateral motion and induce the assembly into planar arrays of cells on semiconductor surfaces in response to temporally and spatially varying electric fields and to projected patterns of illumination.

[0001] This application is a continuation of U.S. Ser. No. 09/320,274,filed on May 28, 1999, which is a continuation-in-part of U.S. Ser. No.09/171,550, filed on Oct. 26, 1998, corresponding to PCT InternationalApplication No. PCT/US97/08159, filed on Apr. 24, 1997, which in turn isbased on U.S. Provisional Application Serial No. 60/016,642, filed onApr. 25, 1997. Applicants hereby claim the priority of these priorapplications pursuant to 35 U.S.C. §§ 119 and 120. These priorapplications are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention generally relates to the field of materialsscience and analytical chemistry.

[0003] The present invention specifically relates to the realization ofa complete, functionally integrated system for the implementation ofbiochemical analysis in a planar, miniaturized format on the surface ofa conductive and/or photoconductive substrate, with applications inpharmaceutical and agricultural drug discovery and in in-vitro orgenomic diagnostics. In addition, the method and apparatus of thepresent invention may be used to create material surfaces exhibitingdesirable topographical relief and chemical functionality, and tofabricate surface-mounted optical elements such as lens arrays.

BACKGROUND OF THE INVENTION

[0004] I—Ions, Electric Fields and Fluid Flow: Field-induced Formationof Planar Bead Arrays

[0005] Electrokinesis refers to a class of phenomena elicited by theaction of an electric field on the mobile ions surrounding chargedobjects in an electrolyte solution. When an object of given surfacecharge is immersed in a solution containing ions, a diffuse ion cloudforms to screen the object's surface charge. This arrangement of a layerof (immobile) charges associated with an immersed object and thescreening cloud of (mobile) counter-ions in solution is referred to as a“double layer”. In this region of small but finite thickness, the fluidis not electroneutral. Consequently, electric fields acting on thisregion will set in motion ions in the diffuse layer, and these will inturn entrain the surrounding fluid. The resulting flow fields reflectthe spatial distribution of ionic current in the fluid. Electroosmosisrepresents the simplest example of an electrokinetic phenomenon. Itarises when an electric field is applied parallel to the surface of asample container or electrode exhibiting fixed surface charges, as inthe case of a silicon oxide electrode (in the range of neutral pH). Ascounter-ions in the electrode double layer are accelerated by theelectric field, they drag along solvent molecules and set up bulk fluidflow. This effect can be very substantial in narrow capillaries and maybe used to advantage to devise fluid pumping systems.

[0006] Electrophoresis is a related phenomenon which refers to thefield-induced transport of charged particles immersed in an electrolyte.As with electroosmosis, an electric field accelerates mobile ions in thedouble layer of the particle. If, in contrast to the earlier case, theparticle itself is mobile, it will compensate for this field-inducedmotion of ions (and the resulting ionic current) by moving in theopposite direction. Electrophoresis plays an important role inindustrial coating processes and, along with electroosmosis, it is ofparticular interest in connection with the development of capillaryelectrophoresis into a mainstay of modern bioanalytical separationtechnology.

[0007] In confined geometries, such as that of a shallow experimentalchamber in the form of a “sandwich” of two planar electrodes, thesurface charge distribution and topography of the bounding electrodesurfaces play a particularly important role in determining the natureand spatial structure of electroosmotic flow. Such a “sandwich”electrochemical cell may be formed by a pair of electrodes separated bya shallow gap. Typically, the bottom electrode will be formed by anoxide-capped silicon wafer, while the other electrode is formed byoptically transparent, conducting indium tin oxide (ITO). The silicon(Si) wafer represents a thin slice of a single crystal of silicon whichis doped to attain suitable levels of electrical conductivity andinsulated from the electrolyte solution by a thin layer of silicon oxide(SiOx).

[0008] The reversible aggregation of beads into planar aggregatesadjacent to an electrode surface may be induced by a (DC or AC) electricfield that is applied normal to the electrode surface. While thephenomenon has been previously observed in a cell formed by a pair ofconductive ITO electrodes (Richetti, Prost and Barois, J. PhysiqueLettr. 45, L-1137 through L-1134 (1984)), the contents of which areincorporated herein by reference, it has been only recently demonstratedthat the underlying attractive interaction between beads is mediated byelectrokinetic flow (Yeh, Seul and Shraiman, “Assembly of OrderedColloidal Aggregates by Electric Field Induced Fluid Flow”, Nature 386,57-59 (1997), the contents of which are incorporated herein byreference). This flow reflects the action of lateral non-uniformities inthe spatial distribution of the current in the vicinity of theelectrode. In the simplest case, such non-uniformities are introduced bythe very presence of a colloidal bead near the electrode as a result ofthe fact that each bead interferes with the motion of ions in theelectrolyte. Thus, it has been observed that an individual bead, whenplaced near the electrode surface, generates a toroidal flow of fluidcentered on the bead. Spatial non-uniformities in the properties of theelectrode can also be introduced deliberately by several methods toproduce lateral fluid flow toward regions of low impedance. Thesemethods are described in subsequent sections below.

[0009] Particles embedded in the electrokinetic flow are advectedregardless of their specific chemical or biological nature, whilesimultaneously altering the flow field. As a result, the electricfield-induced assembly of planar aggregates and arrays applies todiverse colloidal particles including: beaded polymer resins (“beads”),lipid vesicles, whole chromosomes, cells and biomolecules includingproteins and DNA, as well as metal or semiconductor colloids andclusters.

[0010] Important for the applications to be described is the fact thatthe flow-mediated attractive interaction between beads extends todistances far exceeding the characteristic bead dimension. Planaraggregates are formed in response to an externally applied electricfield and disassemble when the field is removed. The strength of theapplied field determines the strength of the attractive interaction thatunderlies the array assembly process and thereby selects the specificarrangement adopted by the beads within the array. That is, as afunction of increasing applied voltage, beads first form planaraggregates in which particles are mobile and loosely packed, then assumea tighter packing, and finally exhibit a spatial arrangement in the formof a crystalline, or ordered, array resembling a raft of bubbles. Thesequence of transitions between states of increasing internal order isreversible, including complete disassembly of planar aggregates when theapplied voltage is removed. In another arrangement, at low initialconcentration, beads form small clusters which in turn assume positionswithin an ordered “superstructure”.

[0011] II—Patterning of Silicon Oxide Electrode Surfaces

[0012] Electrode patterning in accordance with a predetermined designfacilitates the quasi-permanent modification of the electrical impedanceof the EIS (Electrolyte-Insulator-Semiconductor) structure of interesthere. By spatially modulating the EIS impedance, electrode-patterningdetermines the ionic current in the vicinity of the electrode. Dependingon the frequency of the applied electric field, beads either seek out,or avoid, regions of high ionic current. Spatial patterning thereforeconveys explicit external control over the placement and shape of beadarrays.

[0013] While patterning may be achieved in many ways, two proceduresoffer particular advantages. First, UV-mediated re-growth of a thinoxide layer on a properly prepared silicon surface is a convenientmethodology that avoids photolithographic resist patterning and etching.In the presence of oxygen, UV illumination mediates the conversion ofexposed silicon into oxide. Specifically, the thickness of the oxidelayer depends on the exposure time and may thus be spatially modulatedby placing patterned masks into the UV illumination path. Thismodulation in thickness, with typical variations of approximately 10Angstroms, translates into spatial modulations in the impedance of theSi/SiOx interface while leaving a flat and chemically homogeneous topsurface exposed to the electrolyte solution. Second. spatial modulationsin the distribution of the electrode surface charge may be produced byUV-mediated photochemical oxidation of a suitable chemical species thatis first deposited as a monolayer film on the SiOx surface. This methodpermits fine control over local features of the electrode double layerand thus over the electrokinetic flow.

[0014] A variation of this photochemical modulation is the creation oflateral gradients in the EIS impedance and hence in the currentgenerated in response to the applied electric field. For example, thisis readily accomplished by controlling the UV exposure so as tointroduce a slow lateral variation in the oxide thickness or in thesurface charge density. As discussed below, control over lateralgradients serves to induce lateral bead transport and facilitates theimplementation of such fundamental operations as capturing andchanneling of beads to a predetermined destination along conduits in theform of impedance features embedded in the Si/SiOx interface.Photochemical patterning of functionalized chemical overlayers alsoapplies to other types of electrode surfaces including ITO.

[0015] III—Light-controlled Modulation of the Interfacial Impedance

[0016] The spatial and temporal modulation of the EIS-impedance inaccordance with a pattern of external illumination provides the basis tocontrol the electrokinetic forces that mediate bead aggregation. Thelight-modulated electrokinetic assembly of planar colloidal arraysfacilitates remote interactive control over the formation, placement andrearrangement of bead arrays in response to corresponding illuminationpatterns and thereby offers a wide range of interactive manipulations ofcolloidal beads and biomolecules.

[0017] To understand the principle of this methodology, it will behelpful to briefly review pertinent photoelectric properties ofsemiconductors, or more specifically, those of the EIS structure formedby the Electrolyte solution (E), the Insulating SiOx layer (I) and theSemiconductor (S). The photoelectric characteristics of this structureare closely related to those of a standard Metal-Insulator-Semiconductor(MIS) or Metal-Oxide-Semiconductor (MOS) devices which are described inS. M. Sze, “The Physics of Semiconductors”, 2nd Edition, Chapt. 7 (WileyInterscience 1981), the contents of which are incorporated herein byreference.

[0018] The interface between the semiconductor and the insulating oxidelayer deserves special attention. Crucial to the understanding of theelectrical response of the MOS structure to light is the concept of aspace charge region of small but finite thickness that forms at theSi/SiOx interface in the presence of a bias potential. In the case ofthe EIS structure, an effective bias, in the form of a junctionpotential, is present under all but very special conditions. The spacecharge region forms in response to the distortion of the semiconductor'svalence and conduction bands (“band bending”) in the vicinity of theinterface. This condition in turn reflects the fact that, while there isa bias potential across the interface, there is ideally no chargetransfer in the presence of the insulating oxide. That is, inelectrochemical language, the EIS structure eliminates Faradaic effects.Instead, charges of opposite sign accumulate on either side of theinsulating oxide layer and generate a finite polarization.

[0019] In the presence of a reverse bias, the valence and conductionband edges of an n-doped semiconductor bend upward near the Si/SiOxinterface and electrons flow out of the interfacial region in responseto the corresponding potential gradient. As a result, a majority carrierdepletion layer is formed in the vicinity of the Si/SiOx interface.Light absorption in the semiconductor provides a mechanism to createelectron-hole pairs within this region. Provided that they do notinstantaneously recombine, electron-hole pairs are split by the locallyacting electric field, and a corresponding photocurrent flows. It isthis latter effect that affords control over the electrokinetic assemblyof beads in the electrolyte solution.

[0020] To understand in more detail the pertinent frequency dependenceof the light-induced modulation of the EIS impedance, two aspects of theequivalent circuit representing the EIS structure are noteworthy. First,there are close analogies between the detailed electricalcharacteristics of the electric double layer at the electrolyte-oxideinterface, and the depletion layer at the interface between thesemiconductor and the insulator. As with the double layer, the depletionlayer exhibits electrical characteristics similar to those of acapacitor with a voltage-dependent capacitance. As discussed,illumination serves to lower the impedance of the depletion layer.Second, given its capacitive electrical response, the oxide layer willpass current only above a characteristic (“threshold”) frequency.Consequently, provided that the frequency of the applied voltage exceedsthe threshold, illumination can lower the effective impedance of theentire EIS structure.

[0021] This effective reduction of the EIS impedance also depends on thelight intensity which determines the rate of generation of electron-holepairs. In the absence of significant recombination, the majority ofphotogenerated electrons flow out of the depletion region and contributeto the photocurrent. The remaining hole charge accumulates near theSi/SiOx interface and screens the electric field acting in the depletionregion. As a result, the rate of recombination increases, and theefficiency of electron-hole separation, and hence the photocurrent,decreases. For given values of frequency and amplitude of the appliedvoltage, one therefore expects that as the illumination intensityincreases, the current initially increases to a maximum level and thendecreases. Similarly, the impedance initially decreases to a minimumvalue (at maximum current) and then decreases.

[0022] This intensity dependence may be used to advantage to induce thelateral displacement of beads between fully exposed and partially maskedregions of the interface. As the illumination intensity is increased,the fully exposed regions will correspond to the regions of interface oflowest impedance, and hence of highest current, and beads will be drawninto these regions. As the fully exposed regions reach the state ofdecreasing photocurrent, the effective EIS impedance in those regionsmay exceed that of partially masked regions, with a resulting inversionof the lateral gradient in current. Beads will then be drawn out of thefully exposed regions. Additionally, time-varying changes in theillumination pattern may be used to effect bead motion.

[0023] IV—Integration of Biochemical Analysis in a Miniaturized, PlanarFormat

[0024] The implementation of assays in a planar array format,particularly in the context of biomolecular screening and medicaldiagnostics, has the advantage of a high degree of parallelity andautomation so as to realize high throughput in complex, multi-stepanalytical protocols. Miniaturization will result in a decrease inpertinent mixing times reflecting the small spatial scale, as well as ina reduction of requisite sample and reagent volumes as well as powerrequirements. The integration of biochemical analytical techniques intoa miniaturized system on the surface of a planar substrate (“chip”)would yield substantial improvements in the performance, and reductionin cost, of analytical and diagnostic procedures.

[0025] Within the context of DNA manipulation and analysis, initialsteps have been taken in this direction (i.e., miniaturization) bycombining on a glass substrate, the restriction enzyme treatment of DNAand the subsequent separation of enzyme digests by capillaryelectrophoresis, see, for example, Ramsey, PCT Publication No. WO96/04547, the contents of which are incorporated herein by reference, orthe amplification of DNA sequences by application of the polymerasechain reaction (PCR) with subsequent electrophoretic separation, see,for example, U.S. Pat. Nos. 5,498,392 and 5,587,128 to Wilding et al.,the contents of which are incorporated herein by reference.

[0026] While these standard laboratory processes have been demonstratedin a miniaturized format, they have not been used to form a completesystem. A complete system will require additional manipulation such asfront-end sample processing, binding and functional assays and thedetection of small signals followed by information processing. The truechallenge is that of complete functional integration because it is herethat system architecture and design constraints on individual componentswill manifest themselves. For example, a fluidic process is required toconcatenate analytical steps that require the spatial separation, andsubsequent transport to new locations, of sets of analyte. Severalpossibilities have been considered including electroosmotic pumping andtransport of droplets by temperature-induced gradients in local surfacetension. While feasible in demonstration experiments, these techniquesplace rather severe requirements on the overall systems lay-out tohandle the very considerable DC voltages required for efficientelectroosmotic mixing or to restrict substrate heating when generatingthermally generated surface tension gradients so as to avoid adverseeffects on protein and other samples.

SUMMARY OF THE INVENTION

[0027] The present invention combines three separate functional elementsto provide a method and apparatus facilitating the real-time,interactive spatial manipulation of colloidal particles (“beads”) andmolecules at an interface between a light sensitive electrode and anelectrolyte solution. The three functional elements are: the electricfield-induced assembly of planar particle arrays at an interface betweenan insulating or a conductive electrode and an electrolyte solution; thespatial modulation of the interfacial impedance by means of UV-mediatedoxide regrowth or surface- chemical patterning; and, finally, thereal-time, interactive control over the state of the interfacialimpedance by light. The capabilities of the present invention originatein the fact that the spatial distribution of ionic currents, and thusthe fluid flow mediating the array assembly, may be adjusted by externalintervention. Of particular interest is the introduction of spatialnon-uniformities in the properties of the pertinent EIS structure. Asdescribed herein, such inhomogeneities, either permanent or temporary innature, may be produced by taking advantage of the physical and chemicalproperties of the EIS structure.

[0028] The invention relates to the realization of a complete,functionally integrated system for the implementation of biochemicalanalysis in a planar, miniaturized format on the surface of a siliconwafer or similar substrate. In addition, the method and apparatus of thepresent invention may be used to create material surfaces exhibitingdesirable topographical relief and chemical functionality, and tofabricate surface-mounted optical elements such as lens arrays.

[0029] The combination of three functional elements endows the presentinvention with a set of operational capabilities to manipulate beads andbead arrays in a planar geometry to allow the implementation ofbiochemical analytical techniques. These fundamental operations apply toaggregates and arrays of colloidal particles including: beaded polymerresins also referred to as latices, vesicles, whole chromosomes, cellsand biomolecules including proteins and DNA, as well as metal orsemiconductor colloids and clusters.

[0030] Sets of colloidal particles may be captured, and arrays may beformed in designated areas on the electrode surface (FIGS. 1a, 1 b andFIGS. 2a-d). Particles, and the arrays they form in response to theapplied field, may be channeled along conduits of any configuration thatare either embedded in the Si/SiOx interface by UV-oxide patterning ordelineated by an external pattern of illumination. This channeling(FIGS. 1c, 1 d, 1 e, FIGS. 3c, 3 d), in a direction normal to that ofthe applied electric field, relies on lateral gradients in the impedanceof the EIS structure and hence in the field-induced current. Asdiscussed herein, such gradients may be introduced by appropriatepatterns of illumination, and this provides the means to implement agated version of translocation (FIG. 1e). The electrokinetic flowmediating the array assembly process may also be exploited for thealignment of elongated particles, such as DNA, near the surface of theelectrode. In addition, the present invention permits the realization ofmethods to sort and separate particles.

[0031] Arrays of colloidal particles may be placed in designated areasand confined there until released or disassembled. The overall shape ofthe array may be delineated by UV-oxide patterning or, in real time, byshaping the pattern of illumination. This capability enables thedefinition of functionally distinct compartments, permanent ortemporary, on the electrode surface. Arrays may be subjected to changesof shape imposed in real time, and they may be merged with other arrays(FIG. 1f) or split into two or more subarrays or clusters (FIG. 1g,FIGS. 4a, 4 b). In addition, the local state of order of the array aswell as the lateral particle density may be reversibly adjusted by wayof the external electric field or modified by addition of a second,chemically inert bead component.

[0032] The present invention also allows for the combination offundamental operations to develop increasingly complex products andprocesses. Examples given herein describe the implementation ofanalytical procedures essential to a wide range of problems in materialsscience, pharmaceutical drug discovery, genomic mapping and sequencingtechnology. Important to the integration of these and otherfunctionalities in a planar geometry is the capability, provided by thepresent invention, to impose temporary or permanent compartmentalizationin order to spatially isolate concurrent processes or sequential stepsin a protocol and the ability to manipulate sets of particles in amanner permitting the concatenation of analytical procedures that areperformed in different designated areas on the substrate surfaces.

[0033] This invention is for a method and apparatus to direct thelateral motion and induce the assembly into planar arrays of cells onsemiconductor surfaces in response to temporally and spatially varyingelectric fields and to projected patterns of illumination.

[0034] The response of different types of cells to variations in thefrequency and voltage of the applied electric field and to specificpatterns and changing intensity of illumination enables the on-cueassembly of multiple arrays of cells in any desired position on thesubstrate, and further enables the repositioning, disassembly andre-assembly and more generally the reconfiguration and segmentation ofsuch arrays. Time-constant and time-varying illumination patterns aregenerated by an optically programmable illumination pattern generatormay be used and can be based on a graphical representation of thepattern created on a personal computer.

[0035] The novel methodology of image cytometry introduced in thepresent invention relates to the general fields of molecular andcellular biology and has applications in the fields of diagnostics,genetic analysis, cell biology and drug discovery. The image cytometryfacilitates a multiplicity of cell-based biological and biochemicalprocedures based on the quantitative analysis of images recorded fromarray of cells. As with Random Encoded Array Detection (READ) orProgrammable Array Reconfiguration and Segmentation (PARSE), theseassays are performed in a highly parallel format in a small volume ofsample on the surface of a semiconductor wafer.

BRIEF DESCRIPTION OF DRAWINGS

[0036] Other objects, features and advantages of the invention discussedin the above brief explanation will be more clearly understood whentaken together with the following detailed description of an embodimentwhich will be understood as being illustrative only, and theaccompanying drawings reflecting aspects of that embodiment, in which:

[0037]FIGS. 1a-h are illustrations of the fundamental operations forbead manipulation;

[0038]FIGS. 2a and 2 b are photographs illustrating the process ofcapturing particles in designated areas on the substrate surface;

[0039]FIGS. 2c and 2 d are photographs illustrating the process ofexcluding particles from designated areas on the substrate surface;

[0040]FIGS. 3a and 3 b are illustrations of the oxide profile of anSi/SiOx electrode;

[0041]FIGS. 3c and 3 d are photographs of the channeling of particlesalong conduits;

[0042]FIGS. 4a and 4 b are photographs of the splitting of an existingaggregate into small clusters;

[0043]FIG. 5 is a photograph of the lensing action of individualcolloidal beads;

[0044]FIGS. 6a-c are side view illustrations of a layout-preservingtransfer process from a microtiter plate to a planar cell;

[0045]FIG. 7 is a photograph of the inclusion of spacer particles withinbead clusters;

[0046]FIG. 8 is an illustration of binding assay variations;

[0047]FIGS. 9a and 9 b are illustrations of two mechanisms of particlesorting, and FIG. 9c is an illustration of the oxide profile of FIG. 9b;

[0048]FIG. 10 is an illustration of a planar array of bead-anchoredprobe-target complexes;

[0049]FIGS. 11a-11 e are illustrations of DNA stretching in accordancewith the present invention;

[0050]FIG. 12 is an illustration of E. coli cells (grown to log phaseand suspended in 280 mM mannitol solution at the density of 10⁸/ml)which form arrays under electric field. (Bright field image. 25× obj, NA0.45. Electric field: 5V, 200 Hz);

[0051]FIG. 13 is an illustration of the same effect as in FIG. 12, withS. cerevisiae cells (baker's yeast). (Bright field image. 25× obj, NA0.45; electric field: 5V, 400 Hz);

[0052]FIG. 14 is an illustration of E. coli forming diamond shapes thatare projected to the surface by an LCD panel controlled by appropriategraphics software; (20× objective, NA 0.28; Electric field: 10V, 7 kHz);

[0053]FIG. 15, is an illustration of S. cerevisiae responding to twocircular illumination patterns;(20× objective, NA 0.28; Electric field:15 V, 15 kHz)

[0054]FIG. 16 is an illustration of S. cerevisiae responding toillumination, with an arrow indicate the direction cells are moving inor being expelled from the illuminated regions (20× objective, NA 0.28;Electric field: 15 V, 15 kHz);

[0055]FIG. 17 is an illustration of E. Coli cells being repelled fromthe illumination circle(20× objective, NA 0.28; Electric field: 10 V, 7kHz; low substrate doping level (1 Ohm cm-10 Ohm cm));

[0056]FIG. 18 is an illustration of E.coli cells following the expansionof the rectangular illuminated shape (20× objective, NA 0.28; Electricfield: 15 V, 8 kHz);

[0057]FIG. 19 is an illustration of E. coli cells being trapped by thelight stripe at the left and being prevented from moving to the circularregion on the right (20× objective, NA 0.28; Electric field: 15 V, 8kHz);

[0058]FIG. 20 is an illustration of an illuminated region containingboth types of cells being suddenly expanded or “dragged”, with E. colicells (left) responding faster than the larger S. cerevisiae cells(right), resulting in segregation. (Bright field image. 25× obj, NA 0.45Electric field: 5V, 400 Hz);

[0059]FIG. 21 is an illustration of immunophenotyping according to thepresent invention;

[0060]FIG. 22 is an illustration of affinity cell detection according tothe present invention;

[0061]FIG. 23 is an illustration of cell sorting and isolation accordingto the present invention;

[0062]FIG. 24 is an illustration of cell-based screening according tothe present invention;

[0063]FIG. 25 is an illustration of cell-secretion based screeningaccording to the present invention;

[0064]FIG. 26 is an illustration of cell to cell interaction accordingto the present invention; and

[0065]FIG. 27 is an illustration of chemotaxis analysis according to thepresent invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] The three functional elements of the present invention may becombined so as to provide a set of fundamental operations for theinteractive spatial manipulation of colloidal particles and molecules,assembled into planar aggregates adjacent to an electrode surface. Inthe following description, fundamental operations in this “toolset” aredescribed in order of increasing complexity. Specifically, it is usefulto adopt a classification scheme based on the total number of inputs andoutputs, or “terminals”, involved in a given operation. For example, themerging of two separate arrays, or sets of particles, into one would bea “three-terminal” operation, involving two inputs and one output. Theconverse three-terminal operation, involving one input and two outputs,is the splitting of a given array into two subarrays.

[0067] Experimental conditions yielding the phenomena depicted in thevarious photo-graphs included herein are as follows. An electrochemicalcell is formed by a pair of planar ITO electrodes, composed of an ITOlayer deposited on a glass substrate, or by a Si/SiOx electrode on thebottom and an ITO electrode on the top, separated by a typical gap of 50microns or less. Given its dependence on the photoelectric properties ofthe Si/SiOx interface, light control is predicated on the use of aSi/SiOx electrode. Leads, in the form of platinum wires, are attached tothe ITO and to the silicon electrode, which is first etched to removethe insulating oxide in the contact region, by means of silver epoxy.The cell is first assembled and then filled, relying on capillaryaction, with a suspension of colloidal beads, 1 or 2 microns indiameter, at a typical concentration of 0.1% solids in 0.1 mM azidesolution, corresponding to approximately 2×10^ 8 particles permilliliter. The number is chosen so as to yield between ½ and 1 fullmonolayer of particles on the electrode surface. Anionic (e.g.,carboxylated polystyrene, silica), cationic (e.g., aminated polystyrene)or nominally neutral (e.g., polystyrene) have all been used todemonstrate the basic phenomena underlying the three functional elementsof the present invention. The silicon electrode was fabricated from a 1inch-square portion of a Si (100) wafer, typically 200-250 micronsthick, n-doped to typically 0.01 Ohm cm resistivity, and capped with athin oxide of typically 30-40 Angstroms thickness. A thick oxide layerof typically 6000-8000 Angstrom thickness, grown under standardconditions in a furnace at 950 degrees C., may be etched by standardphotolithography to define the structures of interest. Alternatively, athin oxide layer may be regrown on a previously stripped surface of(100)-orientation under UV illumination. Given its ease ofimplementation and execution, UV-mediated oxide regrowth is thepreferable technique: it provides the means to pattern the surface byplacing a quartz mask representing the desired pattern in the path of UVillumination and it leaves a chemically homogeneous, topographicallyflat top surface. To avoid non-specific particle adsorption to theelectrode surface, stringent conditions of cleanliness should befollowed, such as those set forth in the General Experimental Conditionsbelow.

[0068] The fundamental one-terminal operation is a “capture-and-hold”operation (FIG. 1a) which forms an array of particles in a designatedarea of arbitrary outline on the surface that is delineated byUV-mediated oxide patterning or by a corresponding pattern ofillumination projected on an otherwise uniform Si/SiOx substratesurface. FIGS. 2a and 2 b illustrate bead capture on a surfacecharacterized by a very thin oxide region 22 (approximately 20-30Angstroms in thickness) and correspondingly low impedance, while theremaining surface is covered with the original, thick oxide withcorrespondingly high impedance. In FIG. 2a, there is no applied field,and hence, no bead capture. In contrast, in FIG. 2b, an electric fieldis applied (10 Vp-p source, 1 kHz) and bead capture occurs within thethin oxide region 22. Under these conditions, an array starts to growwithin less than a second and continues to grow over the nextapproximately 10 seconds as beads arrive from increasingly largerdistances to add to the outward growing perimeter of region 22. Growthstops when the array approaches the outer limit of the delineated targetarea, i.e., the area defined by the thin oxide having a low impedance.The internal state of order of the captured aggregate of beads isdetermined by the strength of the applied voltage, higher valuesfavoring increasingly denser packing of beads and the eventual formationof ordered arrays displaying a hexagonally crystalline configuration inthe form of a bubble raft. The array remains in place as long as theapplied voltage is present. Removal of the applied voltage results inthe disassembly of the array.

[0069] The “capture-and-hold” operation may also be implemented underillumination with visible or infrared light, for example by simplyprojecting a mask patterned with the desired layout onto the Si/SiOxelectrode. A regular 100W quartz microscope illuminator has been usedfor this purpose on a Zeiss UEM microscope, with apertures or masksinserted in the intermediate image plane to provide the required shapein the plane of the electrode (when focused properly under conditions ofKoehler illumination). Alternatively, an IR laser diode with output of 3mW at 650-680 nm also has been used. The use of external illuminationrather than oxide patterning for the spatial confinement of particlesallows the confinement pattern to be easily modified.

[0070] Related to “capture-and-hold” is the one-terminal operation of“exclude-and-hold” (FIG. 1b ) which clears particles from a designatedarea on the surface. Increasing the frequency of the applied voltage toapproximately 100 kHz leads to an inversion in the preference ofparticles which assemble in the thin-oxide portion of the surface (e.g.,region 22, FIG. 2b) and instead form structures decorating the outsideof the target area perimeter. Rather than relying on this effect, theexclusion of particles from the desired areas is also accomplished, inanalogy to the original “capture-and-hold” operations, by simplyembedding the corresponding structure in the Si/SiOx interface byUV-mediated oxide regrowth. In the example of FIGS. 2c and 2 d, this isachieved, under conditions otherwise identical to those described above,with respect to FIGS. 2a and 2 b, by applying 20V (pp) at 10 kHz. Whilethe oxide thickness in the non designated areas 24 is approximately 30Angstroms, the value in the designated square areas 26 is approximately40 Angstroms, implying a correspondingly higher impedance at the appliedfrequency.

[0071] The “capture-and-hold” operation enables the spatialcompartmentalization of the substrate surface into functionally distinctregions. For example, particles of distinct chemical type, introducedinto the electrochemical cell at different times or injected indifferent locations, can be kept in spatially isolated locations byutilizing this operation.

[0072] The fundamental two-terminal operation is translocation (FIG.1c), or the controlled transport of a set of particles from location Oto location F on the surface; here, O and F are target areas to whichthe above-described one-terminal operations may be applied. Theone-dimensional, lateral bead transport used in translocation isachieved by imposing a lateral current along a conduit connecting areasO and F, as shown in FIGS. 3a and 3 b or by projecting a correspondinglinear pattern of illumination. In this channeling operation, beads movein the direction of lower impedance in the direction of the arrow shownin FIGS. 3a and 3 b, in accordance with the underlying electrokineticflow.

[0073] Oxide patterning may be utilized in two ways to create a lateralcurrent along the Si/SiOx interface. The simplest method is depicted inFIG. 3c and shows a large open holding area 32 fed by three narrowconduits 34 defined by etching a thermal oxide. Beads move to theholding area 32 along the narrow conduits 34 to form a bead array. FIG.3d is a large scale view of the array of FIG. 3c. The principle invokedin creating transport is that of changing the aspect ratio (narrowconduit connected to wide holding area) of the embedded pattern withconstant values of thin oxide thickness inside and thick oxide outside,as illustrated in FIG. 3a. In FIGS. 3c and 3 d, the applied voltage was10V (pp) at 10 kHz. An alternative approach for creating bead transport,enabled by UV-mediated oxide regrowth, is to vary the oxide thicknessalong the conduit in a controlled fashion. This is readily accomplishedby UV exposure through a graduated filter. Differences in the oxidethickness between O and F of as little as 5-10 Angstroms suffice toeffect lateral transport. In this situation, the aspect ratio of theconduit and holding areas need not be altered. This is illustrated inFIG. 3b.

[0074] The use of external illumination to define conduits, by varyingthe illumination intensity along the conduit to create the requisiteimpedance gradient, has the advantage that the conduit is only atemporary structure, and that the direction of motion may be modified orreversed if so desired. The present invention provides for mechanisms oflight-mediated active linear transport of planar aggregates of beadsunder interactive control. This is achieved by adjusting an externalpattern of illumination in real time, either by moving the patternacross the substrate surface in such a way as to entrain the illuminatedbead array or by electronically modulating the shape of the pattern toinduce motion of particles.

[0075] Two modes of light-mediated, active transport are:

[0076] i) Direct Translocation (“tractor beam”) which is a method oftranslocating arrays and of delineating their overall shape by adjustingparameters so as to favor particle assembly within illuminated areas ofthe surface, as described herein. Arrays simply follow the imposedpattern. The rate of motion is limited by the mobility of particles inthe fluid and thus depends on particle diameter and fluid viscosity.

[0077] ii) Transverse Array Constriction is a bead transport mechanismrelated to peristaltic pumping of fluids through flexible tubing. Thelight-control component of the present invention may be used for asimple implementation of this very general concept. A multi-componentplanar aggregate of beads is confined to a rectangular channel, byUV-patterning if so desired, or simply by light. Beads are free to movealong the channel by diffusion (in either direction). An illuminationpattern matching the transverse channel dimension is set up and is thenvaried in time so as to produce a transverse constriction wave thattravels in one direction along the channel. Such a constriction wave maybe set up in several ways. A conceptually simple method is to project aconstricting mask onto the sample and move the projected mask pattern inthe desired fashion. This method also may be implemented electronicallyby controlling the illumination pattern of a suitable array of lightsources, thus obviating the need for moving parts in the optical train.

[0078] The control of lateral bead transport by changing or movingpatterns of illumination has the advantage that it may be appliedwhenever and wherever (on a given substrate surface) required, withoutthe need to impose gradients in impedance by predefined UV patterning.On the other hand, a predefined impedance pattern can provide additionalcapabilities in conjunction with light-control. For example, it may bedesirable to transport beads against a substrate-embedded impedancegradient to separate beads on the basis of mobility.

[0079] Conduits connecting O and F need not be straight: as with tracksdirecting the motion of trains, conduits may be shaped in any desirablefashion (FIG. 1d). A gated version of translocation (FIG. 1e) permitsthe transport of particles from O to F only after the conduit is opened(or formed in real time) by a gating signal. This operation utilizes UVoxide patterning to establish two holding areas, O and F, and also lightcontrol to temporarily establish a conduit connecting O and F. Analternative implementation is based on an oxide embedded impedancegradient. A zone along the conduit is illuminated with sufficiently highintensity to keep out particles, thereby blocking the passage. Removal(or reduction in intensity) of the illumination opens the conduit. Inthe former case, light enables the transport of beads, while in thelatter case, light prevents the transport of beads.

[0080] The fundamental three-terminal operations are the merging andsplitting of sets or arrays of beads (FIGS. 1f and 1 g). The merging oftwo arrays (FIG. 1f) involves the previous two fundamental operations of“capture-and-hold”, applied to two spatially isolated sets of beads inlocations O1 and O2, and their respective channeling along mergingconduits into a common target area, and their eventual channeling,subsequent to mixing, or a chemical reaction, into the finaldestination, a third holding area, F. This is accomplished, under theconditions stated above, by invoking one-terminal and gated two-terminaloperations.

[0081] The splitting of an array into two subarrays (FIG. 1g) is aspecial case of a generally more complex sorting operation. Sortinginvolves the classification of beads in a given set or array into one oftwo subsets, for example according to their fluorescence intensity. Inthe simpler special case, a given array, held in area O, is to be splitinto two subarrays along a demarcation line, and subarrays are to bemoved to target areas F1 and F2. Under the conditions stated above, thisis accomplished by applying the “capture-and-hold” operation to thearray in O. Conduits connect O to F1 and F2. High intensity illuminationalong a narrowly focused line serves to divide the array in a definedfashion, again relying on gated translocation to control transport alongconduits away from the holding area O. An even simpler version, termedindiscriminate splitting, randomly assigns particles into F1 and F2 bygated translocation of the array in O into F1 and F2 after conduits areopened as described above.

[0082]FIGS. 4a and 4 b show a variant in which beads in region O (FIG.4a) are split into multiple regions F1, F2, . . . Fn (FIG. 4b). Thisreversible splitting of an aggregate or array into n subarrays, orclusters, is accomplished, for carboxylated polystyrene spheres of 2micron diameter at a concentration corresponding to an electrodecoverage of a small fraction of a monolayer, at a frequency of 500 Hz,by raising the applied voltage from typically 5V (pp) to 20V (pp). Thisfragmentation of an array into smaller clusters reflects the effect of afield-induced particle polarization. The splitting is useful todistribute particles in an array over a wider area of substrate forpresentation to possible analytes in solution, and for subsequentscanning of the individual clusters with analytical instruments to makeindividual readings.

[0083] The three functional elements of the present invention describedherein may be also combined to yield additional fundamental operationsto control the orientation of anisotropic objects embedded in theelectroosmotic flow created by the applied electric field at theelectrode surface. The direction of the flow, in the plane of thesubstrate, is controlled by gradients in the impedance that are shapedin the manner described in connection with the channeling operation.This is used to controllably align anisotropic objects as illustrated inFIG. 1h, and may be applied to stretch out and align biomolecules, suchas DNA.

[0084] An additional fundamental operation that complements the previousset is that of permanently anchoring an array to the substrate. This isbest accomplished by invoking anchoring chemistries analogous to thoserelying on heterobifunctional cross-linking agents invoked to anchorproteins via amide bond formation. Molecular recognition, for examplebetween biotinylated particles and surface-anchored streptavidin,provides another class of coupling chemistries for permanent anchoring.

[0085] General Experimental Conditions

[0086] The functional elements, namely the electric-field inducedassembly of planar particle arrays, the spatial modulation of theinterfacial impedance by means of UV-mediated oxide or surface-chemicalpatterning and finally, the control over the state of the interfacialimpedance by light which are used in the present invention, have beendemonstrated in experimental studies. These studies employed n-dopedsilicon wafers (resistivities in the range of 0.01 Ohm cm), capped witheither thermally grown oxide layers of several thousand Angstromthickness, or with thin oxide layers, regrown after removal of theoriginal “native” oxide in HF, under UV illumination from a deuteriumsource in the presence of oxygen to typical thicknesses between 10 and50 Angstroms. Lithographic patterning of thermally grown oxide employedstandard procedures implemented on a bench top (rather than a cleanroom) to produce features in the range of several microns.

[0087] Surfaces were carefully cleaned in adherence with industrystandard RCA and Piranha cleaning protocols. Substrates were stored inwater produced by a Millipore water purification system prior to use.Surfaces were characterized by measuring the contact angle exhibited bya 20 microliter droplet of water placed on the surface and viewed (fromthe side) through a telescope. The contact angle is defined as the anglesubtended by the surface and the tangent to the droplet contour (in sideview) at the point of contact with the surface. For example, a perfectlyhemispherical droplet shape would correspond to a contact angle of 90degrees. Surface chemical derivatization withmercapto-propyl-trimethoxysilane (2% in dry toluene) produced surfacesgiving typical contact angles of 70 degrees. Oxidation of the terminalthiol functionality under UV irradiation in the presence of oxygenreduced the contact angle to zero in less than 10 min of exposure to UVfrom the deuterium source. Other silane reagents were used in a similarmanner to produce hydrophobic surfaces, characterized by contact anglesin excess of 110 degrees.

[0088] Simple “sandwich” electrochemical cells were constructed byemploying kapton film as a spacer between Si/SiOx and conductive indiumtin oxide (ITO), deposited on a thin glass substrate. Contacts toplatinum leads were made with silver epoxy directly to the top of theITO electrode and to the (oxide-stripped) backside of the Si electrode.In this two-electrode configuration, AC fields were produced by afunction generator, with applied voltages ranging up to 20V andfrequencies varying from DC to 1 MHZ, high frequencies favoring theformation of particle chains connecting the electrodes. Currents weremonitored with a potentiostat and displayed on an oscilloscope. Forconvenience, epi-fluorescence as well as reflection differentialinterference contrast microscopy employed laser illumination.Light-induced modulations in EIS impedance were also produced with asimple 100W microscope illuminator as well as with a 3 mW laser diodeemitting light at 650-680 nm.

[0089] Colloidal beads, both anionic and cationic as well as nominallyneutral, with a diameter in the range from several hundred Angstroms to20 microns, stored in a NaN₂ solution, were employed.

[0090] Close attention was paid to colloidal stability to avoidnon-specific interactions between particles and between particles andthe electrode surface. Bacterial contamination of colloidal suspensionswas scrupulously avoided.

[0091] Typical operating conditions producing, unless otherwiseindicated, most of the results described herein, were: 0.2 mM NaN₂(sodium azide) solutions, containing particles at a concentration so asto produce not more than a complete monolayer of particles whendeposited on the electrode; applied DC potentials in the range of 1-4V,and AC potentials in the range of 1-10V (peak-to-peak) and 500 Hz-10kHz, with an electrode gap of 50 microns; anionic (carboxylatedpolystyrene) beads of 2 micron diameter, as well as (nominally neutral)polystyrene beads of 2-20 micron diameter.

[0092] The method and apparatus of the present invention may be used inseveral different areas, examples of which are discussed in detail. Eachexample includes background information followed by the application ofthe present invention to that particular application.

EXAMPLE I

[0093] Fabrication of Surfaces and Coatings with Designed Properties

[0094] The present invention may be used to fabricate planar surfacesand coatings with designed properties. Specifically, the functionalelements of the present invention enable the formation of arrayscomposed of particles of a wide range of sizes (approximately 100Angstrom to 10 microns) and chemical composition or surfacefunctionality in response to AC or DC electric fields. These arrays maybe placed and delineated in designated areas of the substrate, and theinterparticle spacing and internal state of order within the array maybe controlled by adjusting the applied field prior to anchoring thearray to the substrate. The newly formed surfaces display pre-designedmechanical, optical and chemical characteristics, and they may besubjected to further modification by subsequent treatment such aschemical cross-linking.

[0095] The mechanical and/or chemical modification of surfaces andcoatings principally determines the interaction between materials in awide range of applications that depend on low adhesion (e.g., thefamiliar “non-stick” surfaces important in housewares) or low friction(e.g., to reduce wear in computer hard disks), hydrophobicity (thetendency to repel water, e.g., of certain fabrics), catalytic activityor specific chemical functionality to either suppress molecularinteractions with surfaces or to promote them. The latter area is ofparticular importance to the development of reliable and durablebiosensors and bioelectronic devices. Finally, a large number ofapplications depend on surfaces of defined topography and/or chemicalfunctionality to act as templates controlling the growth morphology ofdeposited materials or as “command surfaces” directing the alignment ofoptically active molecules in deposited thin organic films, as in liquidcrystal display applications.

[0096] Extensive research has been devoted to the formation of surfacesby adsorption of thin organic films of known composition from the liquidor gas phase by several methods. Notwithstanding their seemingsimplicity and wide-spread use, these methods can be difficult to handlein producing reliable and reproducible results. In addition, molecularfilms are not well suited to produce surfaces displaying a regulartopography.

[0097] An alternative approach to the problem is the modification ofconductive surfaces by electrophoretic deposition of suspendedparticulates. This is a widely used technique in industrial settings toproduce paint coatings of metal parts, and to deposit phosphor fordisplay screens. The active deposition process significantly enhancesthe kinetics of formation (in contrast to passive adsorption of organicfilms from solution), an important consideration in practicalapplications. Electrophoretic deposition requires high DC electricfields and produces layers in which particles are permanently adsorbedto the surface. While particles in so-deposited monolayers are usuallyrandomly distributed, the formation of polycrystalline monolayers ofsmall (150 Angstrom) gold colloids on carbon-coated copper grids is alsoknown. However, the use of carbon-coated copper grids as substrates isnot desirable in most applications.

[0098] Prior art methods have been described for the formation ofordered planar arrays of particles under certain conditions. Forexample, the formation of ordered colloidal arrays in response to ACelectric fields on conductive indium tin oxide (ITO) electrodes isknown. However, the resulting layers were composed of small patches ofordered arrays, randomly distributed over the surface of the otherwisebare ITO substrate. Arrays of monodisperse colloidal beads and globularproteins also have been previously fabricated by using convective flowand capillary forces. However, this latter process has the disadvantageof leaving deposited particle arrays immobilized and exposed to air,making it difficult to modify arrays by subsequent liquid phasechemistry.

[0099] The present invention provides a method of forming planar arrayswith precise control over the mechanical, optical and chemicalproperties of the newly created layer. This method has several distinctadvantages over the prior art. These result from the combination of ACelectric field-induced array formation on insulating electrodes(Si/SiOx) that are patterned by UV-mediated oxide regrowth. The processof the present invention enables the formation of ordered planar arraysfrom the liquid phase (in which particles are originally suspended) indesignated positions, and in accordance with a given overall outline.This eliminates the above-stated disadvantages of the prior art, i.e.,dry state, irregular or no topography, random placement within anaggregate, immobilization of particles and uncontrolled, randomplacement of ordered patches on the substrate.

[0100] An advantage of the present invention is that arrays aremaintained by the applied electric field in a liquid environment. Theprocess leaves the array in a state that may be readily disassembled,subjected to further chemical modification such as cross-linking, ormade permanent by chemical anchoring to the substrate. Furthermore, theliquid environment is favorable to ensure the proper functioning of manyproteins and protein supramolecular assemblies of which arrays may becomposed. It also facilitates the subsequent liquid-phase deposition ofadditional layers of molecules (by chemical binding to beads or proteinsin the deposited layer), the cycling of arrays between states ofdifferent density and internal order (including complete disassembly ofthe array) in response to electric fields and the chemical cross-linkingof particles into two-dimensionally connected layers, or gels, formed,for example, of chemically functionalized silica spheres. The presentinvention can be practiced on insulating electrodes such as oxide-cappedsilicon, to minimize Faradaic processes that might adversely affectchemical reactions involved in the gelation process or in anchoring thearray to the substrate. The use of Si/SiOx electrodes also enables thecontrol of array placement by external illumination.

[0101] The formation of colloidal arrays composed of small particles inaccordance with the present invention provides a route to thefabrication of surfaces with relief structure on the scale of theparticle diameter. Aside from their optical properties, such“micro-rough” surfaces are of interest as substrates for the depositionof DNA in such a way as to alleviate steric constraints and thus tofacilitate enzyme access.

[0102] Particles to which the invention applies include silica spheres,polymer colloids, lipid vesicles (and related assemblies) containingmembrane proteins such as bacteriorhodopsin (bR)⁻ a light-driven protonpump that can be extracted in the form of membrane patches and disks orvesicles. Structured and functionalized surfaces composed of photoactivepigments are of interest in the context of providing elements of planaroptical devices for the development of innovative display and memorytechnology. Other areas of potential impact of topographicallystructured and chemically functionalized surfaces are the fabrication oftemplate surfaces for the controlled nucleation of deposited layergrowth and command surfaces for liquid crystal alignment. The presentinvention also enables the fabrication of randomly heterogeneouscomposite surfaces. For example, the formation of arrays composed of amixture of hydrophobic and hydrophilic beads of the same size creates asurface whose wetting and lubrication characteristics may be controlledby the composition of the deposited mixed bead array. In this way, thelocation of the individual beads is random, but the relative proportionof each type of bead within the array is controllable.

EXAMPLE II

[0103] Assembly of Lens Arrays and Optical Diffraction Elements

[0104] The present invention can be used to fabricate lens arrays andother surface-mounted optical elements such as diffraction gratings. Thefunctional elements of the present invention enable the placement anddelineation of these elements on ITO, facilitating integration withexisting planar display technology, and on Si/SiOx, facilitatingintegration with existing silicon-based device technology.

[0105] Silica or other oxide particles, polymer latex beads or otherobjects of high refractive index suspended in an aqueous solution, willrefract light. Ordered planar arrays of beads also diffract visiblelight, generating a characteristic diffraction pattern of sharp spots.This effect forms the basis of holographic techniques in opticalinformation processing applications.

[0106] A.—The present invention provides for the use of arrays ofrefractive colloidal beads as light collection elements in planar arrayformats in conjunction with low light level detection and CCD imaging.CCD and related area detection schemes will benefit from the enhancedlight collection efficiency in solid-phase fluorescence or luminescencebinding assays.

[0107] This assay format relies on the detection of a fluorescencesignal indicating the binding of probes to bead-anchored targets in thevicinity of the detector. To maximize through-put, it is desirable tomonitor simultaneously as many binding events as possible. It is herethat array formation by the methods of the present invention isparticularly valuable because it facilitates the placement and tightpacking of beads in the target area monitored by the CCD detector, whilesimultaneously providing for the additional benefit of lensing actionand the resulting increase in light collection efficiency.

[0108] Increased collection efficiency has been demonstrated inexperiments employing individual, large (10 micron diameter) polystyrenebeads as lensing elements to image small (1 micron diameter) fluorescentpolystyrene beads. Under the experimental conditions set forth above anapplied voltage of 5V (pp) at 300 Hz induced the collection of smallparticles under individual large beads within a second. This is shown inFIG. 5, where small beads alone, e.g., 52, appear dim, whereas smallbeads, e.g., 54, gathered under a large bead 56 appear brighter andmagnified. The small beads redisperse when the voltage is turned off.

[0109] B.—The use of colloidal bead arrays as diffraction gratings andthus as holographic elements is known. Diffraction gratings have theproperty of diffracting light over a narrow range of wavelengths sothat, for given angle of incidence and wavelength of the illuminatinglight, the array will pass only a specific wavelength (or a narrow bandof wave-lengths centered on the nominal value) that is determined by theinter-particle spacing. Widely discussed applications of diffractiongratings range from simple wavelength filtering to the more demandingrealization of spatial filters and related holographic elements that areessential in optical information processing.

[0110] The present invention provides for a rapid and well controlledprocess of forming planar arrays in a state of crystalline order whichwill function as surface-mounted optical diffraction elements. Inaddition, the resulting surfaces may be designed to displaytopographical relief to enhance wave-length selective reflectivity.These arrays may be formed in designated areas on a substrate surface.In contrast to the slow and cumbersome prior art method of fabricatingsuch arrays by way of forming equilibrium crystals in aqueous solutionsof low salt content, the present invention provides a novel approach torapidly and reliably fabricate particle arrays at a solid-liquidinterface. This approach relies on field-induced formation of arrays totrigger the process, and on UV-mediated patterning or light control toposition and shape the arrays. In addition, the inter-particle distance,and internal state of order, and hence the diffraction characteristicsof the array, may be fine-tuned by adjusting the applied electric field.For example, a field-induced, reversible order-disorder transition inthe array will alter the diffraction pattern from one composed of sharpspots to one composed of a diffuse ring. The assembly of such arrays onthe surface of silicon wafers, as described herein, provides a directmethod of integration into existing microelectronic designs. Arrays maybe locked in place by chemical coupling to the substrate surface, or byrelying on van der Waals attraction between beads and substrate.

EXAMPLE III

[0111] A Novel Mechanism for the Realization of a Particle-Based Display

[0112] The present invention provides the elements to implement lateralparticle motion as a novel approach to the realization of aparticle-based display. The elements of the present invention providefor the control of the lateral motion of small particles in the presenceof a pre-formed lens array composed of large, refractive particles.

[0113] Colloidal particulates have been previously employed inflat-panel display technology. The operating principle of these designsis based on electrophoretic motion of pigments in a colored fluidconfined between two planar electrodes. In the OFF (dark) state,pigments are suspended in the fluid, and the color of the fluid definesthe appearance of the display in that state. To attain the ON (bright)state, particles are assembled near the front (transparent) electrodeunder the action of an electric field. In this latter state, incidentlight is reflected by the layer of particles assembled near theelectrode, and the display appears bright. Prototype displays employingsmall reflective particles in accordance with this design are known.However, these displays suffered from a number of serious problemsincluding: electrochemical degradation and lack of colloidal stabilityas a result of prolonged exposure to the high DC electric fieldsrequired to achieve acceptable switching speeds; and non-uniformitiesintroduced by particle migration in response to field gradients inherentin the design of the addressing scheme.

[0114] The present invention provides a novel mechanism for the designof a particle-based display which takes advantage of electricfield-induced array formation as well as controlled, field-inducedlateral particle displacements. First, a lens array composed ofcolloidal beads is formed. This lens array also serves as a spacer arrayto maintain a well-defined gap between the bottom electrode and the topelectrode that may now be placed over the (pre-formed) array. Thisfacilitates fabrication of uniform flat panel displays with a narrow gapthat is determined by the particle diameter.

[0115] Next, small colloidal particles are added to the electrolytesolution in the gap. These may be fluorescent, or may be reflectingincident white light. Under the action of an AC electric field ofappropriate frequency, these small particles can be moved laterally toassemble preferentially within the footprint of a larger bead. Whenviewed through a larger bead, small fluorescent beads assembled under alarge bead appear bright as a result of the increased light collectionefficiency provided by the lensing action of the large bead; this is theON state (FIG. 5). When moved outside the footprint of the larger bead,particles appear dim, and may be made entirely invisible by appropriatemasking; this is the OFF state. The requisite lateral particle motionmay be induced by a change in the applied voltage or a change in lightintensity. Each large or lensing bead introduces a lateral nonuniformityin the current distribution within the electrolyte because the currentis perturbed by the presence of each lensing bead.

[0116] In contrast to the prior art displays, the present inventionemploys AC, not DC fields, and insulating (rather than conductive)electrodes, thereby minimizing electrochemical degradation. The lateralnon-uniformity introduced by the lens array is desirable because itintroduces lateral gradients in the current distribution within thedisplay cell. These gradients mediate the lateral motion of small beadsover short characteristic distances set by the diameter of the largelensing beads, to effect a switching between ON and OFF states. Thus,the present invention readily accommodates existing technology foractive matrix addressing.

EXAMPLE IV

[0117] Separation and Sorting of Beads and Particles

[0118] The present invention can be used to implement several proceduresfor the separation and sorting of colloidal particles and biomoleculesin a planar geometry. Specifically, these include techniques of lateralseparation of beads in mixtures. Individual beads may be removed from anarray formed in response to an electric field by the application ofoptical tweezers.

[0119] The separation of components in a given mixture of chemicalcompounds is a fundamental task of analytical chemistry. Similarly,biochemical analysis frequently calls for the separation ofbiomolecules, beads or cells according to size and/or surface charge byelectrophoretic techniques, while the sorting (most commonly into justtwo sub-classes) of suspended cells or whole chromosomes according tooptical properties such as fluorescence emission is usually performedusing field-flow fractionation including flow cytometry andfluorescence-activated cell sorting.

[0120] In a planar geometry, bead mixtures undergoing diffusion havebeen previously separated according to mobility by application of an ACelectric field in conjunction with lithographic patterning of theelectrode surface designed to promote directional drift. Essentially, asdescribed in U.S. Pat. No. 5,593,565 to Ajdari et al., the contents ofwhich are included herein by reference, the AC or pulsing electric fieldis used to move small beads in a particular direction over a period oftime, advancing beads of higher mobility relative to those of lowermobility. Capillary electrophoresis has been implemented in a planargeometry, see e.g., B. B. Haab and R. A. Mathies, Anal. Chem 67,3253-3260 (1995), the contents of which are incorporated herein byreference.

[0121] The methods of the present invention may be applied in severalways to implement the task of separation, sorting or isolation in aplanar geometry. In contrast to the prior art approaches, the presentinvention provides a significant degree of flexibility in selecting fromamong several available procedures, the one best suited to theparticular task at hand. In some cases, more than one separationtechnique may be applied, and this provides the basis for theimplementation of two-dimensional separation. That is, beads may beseparated according to two different physical-chemical characteristics.For example, beads may first be separated by size and subsequently, byraising the applied frequency to induce chain formation, bypolarizability. This flexibility offers particular advantages in thecontext of integrating analytical functionalities in a planar geometry.Several techniques will now be described.

[0122] i) The present invention may be used to implement “sieving” inlateral, electric field-induced flow on surfaces patterned byUV-mediated oxide regrowth to sort beads in a mixture by size. Thefundamental operations of the invention are invoked to set up directedlateral particle motion along conduits laid out by UV-mediated oxideregrowth. Conduits are designed to contain successively narrowerconstrictions through which particles must pass. Successively finerstages allow only successively smaller particles to pass in this“sieving” mechanism (FIG. 9a). As shown in FIG. 9a, the primary particleflow is in the direction left to right, while a transverse flow isestablished in the top to bottom direction utilizing an oxide profile asshown. Additionally, rows of barriers 92 made from thick oxide arepositioned along the conduit with the spacing between the barriers ineach row decreasing in the transverse direction. As the particles movealong the conduit, the rows of barriers act to separate out smallerparticles in the transverse direction. In contrast to previous methodsbased on electrophoretic separation, large DC electric fields, and theattendant potential problem of electrolysis and interference fromelectroosmotic flow in a direction opposite to the field-directedparticle transport, the present invention uses AC electric fields andlateral gradients in interfacial impedance to produce transport. Thepresent method has the advantage of avoiding electrolysis and it takesexplicit advantage of electroosmotic flow to produce and controlparticle transport.

[0123] In addition, the use of Si/SiOx electrodes enables the use of thelight-control component of the present invention to modify lateraltransport of beads in real time. For example, external illumination maybe employed to locally neutralize the lateral impedance gradient inducedby UV-mediated oxide regrowth. Particles in these neutral “zones” wouldno longer experience any net force and come to rest. This principle maybe used as a basis for the implementation of a scheme to locallyconcentrate particles into sharp bands and thereby to improve resolutionin subsequent separation.

[0124] ii) The present invention may be used to implement “zonerefining”, a process of excluding minority components of a mixture bysize or shape from a growing crystalline array of majority component.This process explicitly depends on the capabilities of the presentinvention to induce directional crystallization.

[0125] The process of zone refining is employed with great success inproducing large single crystals of silicon of very high purity byexcluding impurities from the host lattice. The concept is familiar fromthe standard chemical procedure of purification by re-crystallization inwhich atoms or molecules that are sufficiently different in size, shapeor charge from the host species so as not to fit into the forming hostcrystal lattice as a substitutional impurity, are ejected into solution.

[0126] By enabling the growth of planar arrays, in a given direction andat a controlled rate, the present invention facilitates theimplementation of an analogous zone refining process for planar arrays.The most basic geometry is the linear geometry. A multi-componentmixture of beads of different sizes and/or shapes is first captured in arectangular holding area on the surface, laid out by UV-patterning.Next, crystallization is initiated at one end of the holding area byillumination and allowed to slowly advance across the entire holdingarea in response to an advancing pattern of illumination. In general,differences of approximately 10% in bead radius trigger ejection.

[0127] iii) The present invention may be used to implement fractionationin a transverse flow in a manner that separates particles according tomobility.

[0128] Field-flow fractionation refers to an entire class of techniquesthat are in wide use for the separation of molecules or suspendedparticles. The principle is to separate particles subjected to fluidflow in a field acting transverse to the flow. A category of suchtechniques is subsumed under the heading of electric-field flowfractionation of which free-flow electrophoresis is a pertinent examplebecause it is compatible with a planar geometry. Free-flowelectrophoresis employs the continuous flow of a replenished bufferbetween two narrowly spaced plates in the presence of a DC electricfield that is applied in the plane of the bounding plates transverse tothe direction of fluid flow. As they traverse the electric field,charged particles are deflected in proportion to their electrophoreticmobility and collected in separate outlets for subsequent analysis. Incontrast to conventional electrophoresis, free-flow electrophoresis is acontinuous process with high throughput and it requires no supportingmedium such as a gel.

[0129] The present invention enables the implementation of field-flowfractionation in a planar geometry. As previously discussed herein,impedance gradients imposed by UV-oxide profiling serve to mediateparticle motion along the electrode surface in response to the externalelectric field. In a cell with a narrow gap, the resultingelectrokinetic flow has a “plug” profile and this has the advantage ofexposing all particles to identical values of the flow velocity field,thereby minimizing band distortions introduced by the parabolic velocityprofile of the laminar flow typically employed in free-flowelectrophoresis.

[0130] A second flow field, transverse to the primary flow direction,may be employed to mediate particle separation. This deflecting flow maybe generated in response to a second impedance gradient. A convenientmethod of imposing this second gradient is to take advantage of UV-oxidepatterning to design appropriate flow fields. Both longitudinal andtransverse flow would be recirculating and thus permit continuousoperation even in a closed cell, in contrast to any related prior arttechnique.

[0131] Additional flexibility is afforded by invoking the light-controlcomponent of the present invention to illuminate the substrate with astationary pattern whose intensity profile in the direction transverseto the primary fluid flow is designed to induce the desired impedancegradient and hence produce a transverse fluid flow. (FIG. 9b). This hasthe significant advantage of permitting selective activation of thetransverse flow in response to the detection of a fluorescent beadcrossing a monitoring window upstream. Non-fluorescent beads would notactivate the transverse flow and would not be deflected. This procedurerepresents a planar analog of flow cytometry, or fluorescence-activatedcell sorting.

[0132] iv) The invention may be used to induce the formation of particlechains in the direction normal to the plane of the electrode. The chainsrepresent conduits for current transport between the electrodes andtheir formation may reflect a field-induced polarization. Chains aremuch less mobile in transverse flow than are individual particles sothat this effect may be used to separate particles according to thesurface properties that contribute to the net polarization. The effectof reversible chain formation has been demonstrated under theexperimental conditions stated herein. For example, the reversibleformation of chains occurs, for carboxylated polystyrene beads of 1micron diameter, at a voltage of 15 V (pp) at frequencies in excess of 1MHz.

[0133] v) The invention may be used to isolate individual beads from aplanar array.

[0134] Fluorescence binding assays in a planar array format, asdescribed herein, may produce singular, bright beads within a largearray, indicating particularly strong binding. To isolate and retrievethe corresponding beads, optical tweezers in the form of a sharplyfocused laser spot, may be employed to lock onto an individual bead ofinterest. The light-control component of the present invention may beused in conjunction with the optical tweezers to retrieve such anindividual bead by moving the array relative to the bead, or vice versa,or by disassembling the array and retaining only the marked bead. Thisis a rather unique capability that will be particularly useful in thecontext of isolating beads in certain binding assays.

[0135] Commercial instrumentation is available to position opticaltweezers in the field of a microscope. Larger scale motion isfacilitated by translocating the array in-situ or simply by moving theexternal sample fixture. This process lends itself to automation inconjunction with the use of peak-finding image analysis software andfeedback control.

[0136] vi) The invention may be used to implement a light-induced arraysectioning (“shearing”) operation to separate fluorescent, or otherwisedelineated portions of an array from the remainder. This operation makesit possible to segment a given array and to isolate the correspondingbeads for downstream analysis.

[0137] The basis for the implementation of this array segmentation isthe light-control component of the present invention, in the mode ofdriving particles from an area of a Si/SiOx interface that isilluminated with high intensity. It is emphasized here that this effectis completely unrelated to the light-induced force on beads thatunderlies the action of optical tweezers. The present effect whichoperates on large sets of particles, was demonstrated under theexperimental conditions stated herein using a 100W illuminator on aZeiss UEM microscope operated in epi-illumination. A simpleimplementation is to superimpose, on the uniform illumination patternapplied to the entire array, a line-focussed beam that is positioned bymanipulation of beam steering elements external to the microscope. Beadsare driven out of the illuminated linear portion. Other implementationstake advantage of two separately controlled beams that are partiallysuperimposed. The linear sectioning can be repeated in differentrelative orientations of shear and array.

EXAMPLE V

[0138] Fabrication of Spatially Encoded Bead Arrays

[0139] The present invention provides a method to transfer suspensionsof beads or biomolecules to the electrode surface in such a way as topreserve the spatial encoding in the original arrangement of reservoirs,most commonly the conventional 8×12 arrangement of wells in a microtiterplate. Such a fluid transfer scheme is of significant practicalimportance given that compound libraries are commonly handled andshipped in 8×12 (or equivalent) wells.

[0140] The present invention utilizes chemical patterning to defineindividual compartments for each of M×N sets of beads and confine themaccordingly. In the present instance, patterning is achieved byUV-mediated photochemical oxidation of a monolayer of thiol-terminatedalkylsilane that is chemisorbed to the Si/SiOx substrate. Partialoxidation of thiol moieties produces sulfonate moities and renders theexposed surface charged and hydrophilic. The hydrophilic portions of thesurface, in the form of a grid of squares or circles, will serve asholding areas.

[0141] In accordance with the present invention, the first function ofsurface-chemical patterning into hydrophilic sections surrounded byhydrophobic portions is to ensure that droplets, dispensed fromdifferent wells, will not fuse once they are in contact with thesubstrate. Consequently, respective bead suspensions will remainspatially isolated and preserve the lay-out of the original M×N wellplate. The second role of the surface chemical patterning of the presentinvention is to impose a surface charge distribution, in the form of theM×N grid pattern, which ensures that individual bead arrays will remainconfined to their respective holding areas even as the liquid phasebecomes contiguous.

[0142] The layout-preserving transfer procedure involves the stepsillustrated in FIGS. 6a-c. First, as shown in side view in FIG. 6a, theM×N plate of wells 62 is registered with the pattern 64 on the planarsubstrate surface. Well bottoms 62, are pierced to allow for theformation of pendant drops of suspension or, preferably, the process isfacilitated by a fixture (not shown) providing M×N effective funnels tomatch the geometric dimensions of the M×N plate on the top and reducethe size of the dispensing end. Such a dispensing fixture will alsoensure the precise control of droplet volumes, adjusted so as toslightly overfill the target holding area on the patterned substratesurface. The set of M×N drops is then deposited by bringing them incontact with the hydrophilic holding areas of the pre-patternedsubstrate and relying on capillary action.

[0143] Next, the plate is retracted, and the top electrode is carefullylowered to form the electrochemical cell, first making contact as shownin FIG. 6b, with individual liquid-filled holding areas on the substrateto which suspensions are confined. Overfilling ensures that contact ismade with individual suspensions. The electric field is now turned on toinduce array formation in the M×N holding areas and to ensure thepreservation of the overall configuration of the M×N sets of beads whilethe gap is closed further (or filled with additional buffer) toeventually fuse individual droplets of suspension into a contiguousliquid phase as shown in FIG. 6c. In the fully assembled cell of FIG.6c, while the droplets are fused together, the beads from each dropletare maintained in and isolated in their respective positions, reflectingthe original M×N arrangement of wells. The present invention thusprovides for the operations required in this implementation of alayout-preserving transfer procedure to load planar electrochemicalcells.

EXAMPLE VI

[0144] Fabrication of Dynamic Planar Bead Arrays for Parallel Assays

[0145] The present invention provides a method to produce aheterogeneous panel of beads and potentially of biomolecules forpresentation to analytes in an adjacent liquid. A heterogeneous panelcontains particles or biomolecules which differ in the nature of thechemical or biochemical binding sites they offer to analytes insolution. The present method relies on the functional elements of theinvention to assemble a planar array of a multi-component mixture ofbeads which carry chemical labels in the form of tag molecules and maybe so identified subsequent to performing the assay. In the event ofbinding, the analyte is identified by examination of the bead, orcluster of beads, scoring positive.

[0146] Diagnostic assays are frequently implemented in a planar formatof a heterogeneous panel, composed of simple ligands, proteins and otherbiomolecular targets. For example, in a diagnostic test kit, aheterogeneous panel facilitates the rapid testing of a given analyte,added in solution, against an entire set of targets. Heterogeneouspanels of proteins are of great current interest in connection with theemerging field of proteome research. The objective of this research isto identify, by scanning the panel with sensitive analytical techniquessuch as mass spectrometry, each protein in a multi-component mixtureextracted from a cell and separated by two-dimensional gelelectrophoresis. Ideally, the location of each spot uniquely correspondsto one particular protein. This analysis would permit, for example, thedirect monitoring of gene expression levels in a cell during aparticular point in its cycle or at a given stage during embryonicdevelopment.

[0147] The fabrication of an array of heterogeneous targets is centralto recently proposed strategies of drug screening and DNA mutationanalysis in a planar format. The placement of ligands in a specificconfiguration on the surface of a planar substrate serves to maintain akey to the identity of any one in a large set of targets presentedsimultaneously to an analyte in solution for binding or hybridization.In an assay relying on fluorescence, binding to a specific target willcreate bright spots on the substrate whose spatial coordinates directlyindicate the identity of the target.

[0148] Three principal strategies have been previously employed tofabricate heterogeneous panels. First, protein panels may be created bytwo-dimensional gel electrophoresis, relying on a DC electric field toseparate proteins first by charge and then by size (or molecularweight). Even after many years of refinement, this technique yieldsresults of poor reproducibility which are generally attributed to thepoorly defined properties of the gel matrix.

[0149] Second, individual droplets, drawn from a set of reservoirscontaining solutions of the different targets, may be dispensed eitherby hand or by employing one of several methods of automated dispensing(or “printing”; see e.g., Schena et al., Science 270, 467-470 (1995),the contents of which are incorporated herein by reference). Printinghas been applied to create panels of oligonucleotides intended forscreening assays based on hybridization. Printing leaves a dried sampleand may thus not be suitable for proteins that would denature under suchconditions. In addition, the attendant fluid handling problems inherentin maintaining, and drawing samples from a large number of reservoirsare formidable.

[0150] Third, target ligands may be created by invoking a variant ofsolid phase synthesis based on a combinatorial strategy ofphotochemically activated elongation reactions. This approach has beenlimited by very formidable technical problems in the chemical synthesisof even the simplest, linear oligomers. The synthesis of non-linearcompounds in this planar geometry is extremely difficult.

[0151] The present invention of forming heterogeneous panels requiresthe chemical attachment of target ligands to beads. Ligands may becoupled to beads “off-line” by a variety of well established couplingreactions. For present purposes, the bead identity must be chemicallyencoded so it may be determined as needed. Several methods of encoding,including binary encoding, of beads are available. For example, shortoligonucleotides may serve the purpose of identifying a bead via theirsequence which may be determined by microscale sequencing techniques.Alternatively, chemically inert molecular tags may be employed that arereadily identified by standard analytical techniques.

[0152] In contrast to all prior art methods, the present inventionprovides a novel method to create heterogeneous panels by in-situ,reversible formation of a planar array of chemically encoded beads insolution adjacent to an electrode. The array may be random with respectto chemical identity but is spatially ordered. This procedure offersseveral advantages. First, it is reversible so that the panel may bedisassembled following the binding assay to discard beads scoringnegative. Positive beads may be subjected to additional analysis withoutthe need for intermediate steps of sample retrieval, purification ortransfer between containers. Second, the panel is formed when needed,that is, either prior to performing the actual binding assay, orsubsequent to performing the assay on the surface of individual beads insuspension. The latter mode minimizes potential adverse effects that canarise when probes bind to planar target surfaces with a highconcentration of target sites. Third, to accommodate optical analysis ofindividual beads, interparticle distances within the array may beadjusted by field-induced polarization or by the addition of inertspacer particles that differ in size from the encoded beads. FIG. 7shows the use of small spacer beads 72 for separating encoded beads 74.As shown, the spacing of beads 74 is greater than the spacing ofcomparable beads in FIG. 4b. Finally, UV-mediated oxide regrowth, asprovided by the present invention, readily facilitates the embedding ofa grid pattern of selected dimension into the substrate to ensure theformation of small, layout-preserving subarrays in the low-impedancefields of the grid.

[0153] To create the panel, a multi-component mixture of beads carrying,for example, compounds produced by bead-based combinatorial chemistry,is placed between electrodes. Each type of bead may be present inmultiple copies. Arrays are formed in response to an external field in adesignated area of the electrode surface. This novel approach of in-situassembly of panels relies on beads that carry a unique chemical label,or code, to permit their identification subsequent to the completion ofa binding assay. Alternatively, beads may be marked (“painted”) on-lineby way of a photochemical bead-coloring method. Selected beads in anarray are individually illuminated by a focused light source to triggera coloring reaction on the bead surface or in the bead interior toindicate a positive assay score. Beads so marked can be subsequentlyseparated from unmarked beads by a light-activated sorting methoddescribed herein. Numerous UV-activated reactions are available toimplement this bead-coloring method.

[0154] The present invention provides for several methods of discardingbeads with negative scores, typically the vast majority, while retainingthose with positive scores. This method take advantage of the fact that,in contrast to all prior art methods, the array represents a temporaryconfiguration of particles that is maintained by the applied electricfield and may be rearranged or disassembled at will. This capability,along with the fact that biomolecules are never exposed to air (as inthe prior art method of printing) facilitates the in-situ concatenationof analytical procedures that require the heterogeneous panel inconjunction with subsequent, “downstream” analysis.

[0155] First, if positive beads are clustered in a subsection of thearray, the light-controlled array splitting operation of the presentinvention may be invoked to dissect the array so as to discard negativeportions of the array (or recycle them for subsequent use). Second, ifpositive and negative beads are randomly interspersed, afluorescence-activated sorting method, implemented on the basis of thepresent invention in a planar format, as described herein, may beinvoked. In the case of fluorescence-activated sorting, positive andnegative beads may be identified as bright and dark objects,respectively. In the special case that only a few positive beads standout, these may be removed from the array by locking onto them withoptical tweezers, a tool to trap and/or manipulate individual refractiveparticles under illumination, and disassembling the array by removingthe field, or subjecting the entire array to lateral displacement by thefundamental operations of the present invention.

[0156] The typical task in screening a large set of compounds is one oflooking for a very small number of positive events in a vast number oftests. The set of discarded beads will typically involve the majority ateach stage in the assay. The procedure of the present inventiontherefore minimizes the effort invested in negative events, such as thechallenging in-situ synthesis of target ligands irrespective of whetheror not they will prove to be of interest by binding a probe offered insolution.

[0157] The method of forming a heterogeneous panel according to thepresent invention contains beads of each type in generally randomassembly. The creation of a heterogeneous panel with each position inthe panel containing a cluster of beads of the same type, that is, beadsoriginating in the same reservoir (FIG. 6a), may be desirable so as toensure a sufficiently large number of positive events to facilitatedetection. A practical solution follows from the application of thelayout-preserving fluidic transfer scheme described herein. In thisprocedure, beads from an M×N well plate are transferredlayout-preservingly onto a chemically patterned substrate in such a wayas to preserve the spatial encoding of bead identities.

EXAMPLE VII

[0158] Binding and Functional Assays in Planar Bead Array Format

[0159] The present invention can be used to implement mixed-phasebinding assays as well as certain functional assays in a planar arrayformat. Several combinations are possible reflecting the presence ofprobe or target in solution, on the surface of colloidal beads, or onthe electrode surface. The methods of the present invention facilitatethe formation of a planar array to present targets to probes in solutionprior to performing the binding assay (“pre-formed” array; FIG. 8).Alternatively, a planar array of beads may be formed in front of adetector surface subsequent to performing the binding assay insuspension (“post-formed” array; FIG. 8). The present invention alsoprovides the methods to implement functional assays by enabling theassembly of certain cell types adjacent to a planar detector or sensorsurface to monitor the effects of exposure of the cells to smallmolecule drugs in solution.

[0160] Binding assays, particularly those involving proteins such asenzymes and antibodies, represent a principal tool of medicaldiagnostics. They are based on the specific biochemical interactionbetween a probe, such as a small molecule, and a target, such as aprotein. Assays facilitate the rapid detection of small quantities of ananalyte in solution with high molecular specificity. Many procedureshave been designed to produce signals to indicate binding, eitheryielding a qualitative answer (binding or no binding) or quantitativeresults in the form of binding or association constants. For example,when an enzyme binds an analyte, the resulting catalytic reaction may beused to generate a simple color change to indicate binding, or it may becoupled to other processes to produce chemical or electrical signalsfrom which binding constants are determined. Monoclonal antibodies,raised from a single common precursor, may be prepared to recognizevirtually any given target, and immunoassays, based on antibody-antigenrecognition and binding, have developed into an important diagnostictool. As with enzyme binding, antibody binding of an antigenic analytemay be detected by a variety of techniques including the classic methodof enzyme-linked immunoassays (ELISA) in which the reaction of anantibody-coupled enzyme is exploited as an indicator. A common andconceptually simple scheme ensures the detection of antibody binding toa target analyte by supplying a fluorescently labeled second antibodythat recognizes the first (or primary) antibody.

[0161] Binding assays involving soluble globular proteins are oftenperformed in solution to ensure unbiased interactions between proteinand target. Such liquid phase assays, especially when performed at lowconcentrations of target or probe, minimize potential difficulties thatmay arise when either target or probe are present in abundance or inclose proximity. By the same token, the kinetics tend to be slow.Cooperative effects, such as crowding, arising from the close proximityof probes must be carefully controlled when either probe or target ischemically anchored to a solid substrate.

[0162] Nonetheless, this latter solid phase format of binding assays isalso very commonly employed whenever the situation demands it. Forexample, the presence of a protein on the surface of a cell may beexploited in “panning” for the cells that express this protein in thepresence of many other cells in a culture that do not: desired cellsattach themselves to the surface of a container that is pre-coated witha layer of a secondary antibody directed against a primary antibodydecorating the desired cell-surface protein. Similarly, certain phagesmay be genetically manipulated to display proteins on their surface, andthese may be identified by a binding assay involving a small moleculeprobe such as an antigen if the protein displayed is an antibody (Watsonet al., “Recombinant DNA”, 2nd Edition (Scientific American Books, W. H.Freeman and Co., New York, N.Y., 1983), the contents of which areincorporated herein by reference). In addition, the planar geometryaccommodates a variety of optical and electrical detection schemesimplemented in transducers and sensors.

[0163] A combination of liquid phase and solid phase assay may bedeveloped by using beads that are decorated with either probe or target,as in procedures that employ decorated magnetic beads for samplepreparation or purification by isolating binding from non-bindingmolecules in a given multi-component mixture. Recent examples of the useof these beads include the purification of templates for DNA sequencingapplications or the extraction of mRNAs from (lysed) cells byhybridization to beads that are decorated with poly-adenine (polyA)residues.

[0164] Functional assays involving suitable types of cells are employedto monitor extracellular effects of small molecule drugs on cellmetabolism. Cells are placed in the immediate vicinity of a planarsensor to maximize the local concentration of agents released by thecell or to monitor the local pH.

[0165] The present invention provides the means to implement mixed phasebinding assays in a planar geometry with a degree of flexibility andcontrol that is not available by prior art methods. Thus, it offers theflexibility of forming, in-situ, reversibly and under external spatialcontrol, either a planar panel of target sites for binding of analytepresent in an adjacent liquid phase, or a planar array of probe-targetcomplexes subsequent to performing a binding assay in solution. Bindingmay take place at the surface of individual beads suspended in solution,at the surface of beads pre-assembled into arrays adjacent to theelectrode surface, or at the electrode surface itself. Either the targetor probe molecule must be located on a bead to allow for a bead-basedassay according to the present invention. As shown in FIG. 8, if theprobe molecule P is located on a bead, then the target molecule T may beeither in solution, on a bead or on the electrode surface. The converseis also true.

[0166] For example, the methods of the present invention may be used toimplement panning, practiced to clone cell surface receptors, in a farmore expeditious and controlled manner than is possible by the prior artmethod. Given a substrate that has been coated with a layer of antibodydirected against the sought-after cell surface protein, the presentinvention facilitates the rapid assembly of a planar array of cells ordecorated beads in proximity to the layer of antibodies and thesubsequent disassembly of the array to leave behind only those cells orbeads capable of forming a complex with the surface-bound antibody.

[0167] A further example of interest in this category pertains to phagedisplays. This technique may be employed to present a layer of proteintargets to bead-anchored probes. Bead arrays may now be employed toidentify a protein of interest. That is, beads are decorated with smallmolecule probes and an array is formed adjacent to the phage display.Binding will result in a probe-target complex that retains beads whileothers are removed when the electric field is turned off, or whenlight-control is applied to remove beads from the phage display. Ifbeads are encoded, many binding tests may be carried out in parallelbecause retained beads may be individually identified subsequent tobinding.

[0168] The methods of the present invention readily facilitatecompetitive binding assays. For example, subsequent to binding of afluorescent probe to a target-decorated bead in solution and theformation of a planar bead array adjacent to the electrode, fluorescentareas within the array indicate the position of positive targets, andthese may be further probed by subjecting them to competitive binding.That is, while monitoring the fluorescence of a selected section of theplanar array, an inhibitor (for enzyme assays) or other antagonist (ofknown binding constant) is added to the electrochemical cell, and thedecrease in fluorescence originating from the region of interest ismeasured as a function of antagonist concentration to determine abinding constant for the original probe. This is an example of aconcatenation of analytical steps that is enabled by the methods of thepresent invention.

[0169] The fact that a probe-target complex is fixed to a colloidalbead, as in the methods of the present invention, conveys practicaladvantages because this facilitates separation of positive from negativeevents. Particularly when solid phase assays are performed on a planarsubstrate, an additional advantage of planar bead arrays is theenhancement of light collection efficiency provided by the beads, asdiscussed herein.

[0170] If desired, beads may serve strictly as delivery vehicles forsmall molecule probes. That is, an array of probe-decorated beads isformed adjacent to a target-decorated surface in accordance with themethods of the present invention. UV-activated cleavage of the probefrom the bead support will ensure that the probe is released in closeproximity to the target layer, thereby enhancing speed and efficiency ofthe assay. The identity of the particular probe interacting with thetarget may be ascertained from the positional location of the beaddelivering the probe.

[0171] The methods of the present invention apply not only to colloidalbeads of a wide variety (that need no special preparative procedures tomake them magnetic, for example), but also to lipid vesicles and cellsthat are decorated with, or contain embedded in their outer wall, eitherprobe or target. The methods of the present invention may therefore beapplied not only to bead-anchored soluble proteins but potentially tointegral membrane receptors or to cell surface receptors.

[0172] In particular, the rapid assembly of cells in a designated areaof the substrate surface facilitates the implementation of highlyparallel cell-based functional assays. The present invention makes itpossible to expose cells to small molecule drug candidates in solutionand rapidly assemble them in the vicinity of a sensor embedded in theelectrode surface, or to expose pre-assembled cells to such agents thatare released into the adjacent liquid phase. In the simplest case, allcells will be of the same type, and agents will be administeredsequentially. Even in this sequential version, electrokinetic mixingwill enhance through-put. However, as described herein, the methods ofthe present invention also enable the parallel version of binding assaysand thus of functional assays in a planar format by encoding theidentity of different cells by a “Layout-Preserving Transfer” processfrom an 8×12 well plate, as discussed herein, and to isolate cellsscoring positive by providing feed-back from a spatially resolvedimaging or sensing process to target a specific location in the array ofcells.

EXAMPLE VIII

[0173] Screening for Drug Discovery in Planar Geometry

[0174] The functional elements of the present invention may be combinedto implement procedures for handling and screening of compound andcombinatorial libraries in a planar format. The principal requisiteelements of this task are: sample and reagent delivery from the set oforiginal sample reservoirs, commonly in a format of 8×12 wells in amicrotiter plate, into a planar cell; fabrication of planar arrays oftargets or of probe-target complexes adjacent to the planar electrodesurface prior to or subsequent to performing a binding assay; evaluationof the binding assay by imaging the spatial distribution of markerfluorescence or radioactivity, optionally followed by quantitativepharmacokinetic measurements of affinity or binding constants; isolationof beads scoring positive, and removal from further processing of otherbeads; and collection of specific beads for additional downstreamanalysis. The present invention relates to all of these elements, andthe fundamental operations of the invention provide the means toconcatenate these procedures in a planar format.

[0175] A central issue in the implementation of cost-effectivestrategies for modern therapeutic drug discovery is the design andimplementation of screening assays in a manner facilitating highthroughput while providing pharmacokinetic data as a basis to selectpromising drug leads from a typically vast library of compounds. Thatis, molecular specificity for the target, characterized by a bindingconstant, is an important factor in the evaluation of a new compound asa potential therapeutic agent. Common targets include enzymes andreceptors as well as nucleic acid ligands displaying characteristicsecondary structure.

[0176] The emerging paradigm for lead discovery in pharmaceutical andrelated industries such as agricultural biotechnology, is the assemblyof novel synthetic compound libraries by a broad variety of new methodsof solid state “combinatorial” synthesis. Combinatorial chemistry refersto a category of strategies for the parallel synthesis and testing ofmultiple compounds or compound mixtures in solution or on solidsupports. For example, a combinatorial synthesis of a linearoligopeptide containing n amino acids would simultaneously create allcompounds representing the possible sequence permutations of n aminoacids. The most commonly employed implementation of combinatorialsynthesis relies on colloidal bead supports to encode reaction steps andthus the identity of each compound. Beads preferred in current practicetend to be large (up to 500 microns in diameter) and porous to maximizetheir compound storage capacity, and they must be encoded to preservethe identity of the compound they carry.

[0177] Several methods of encoding, or binary encoding, of beads areavailable. Two examples are as follows. First, beads may be labeled withshort oligonucleotides such as the 17-mers typically employed inhybridization experiments. The sequence of such short probes may bedetermined by microscale sequencing techniques such as directMaxam-Gilbert sequencing or mass spectrometry. This encoding scheme issuitable when the task calls for screening of libraries of nucleic acidligands or oligopeptides. Second, members of a combinatorial library maybe associated with chemically inert molecular tags. In contrast to theprevious case, these tag molecules are not sequentially linked. Instead,the sequence of reaction steps is encoded by the formal assignment of abinary code to individual tag molecules and their mixtures that areattached to the bead in each successive reaction step. The tags arereadily identified by standard analytical techniques such as gaschromatography. This general encoding strategy is currently employed inthe synthesis of combinatorial libraries on colloidal beads.

[0178] Commercial compound libraries are large, given that even for theaforementioned 17-mer, the number of sequence permutations is 4^ 17, orapproximately 10^ 10. However, the high specificity of typicalbiological substrate-target interactions implies that the vast majorityof compounds in the collection will be inactive for any one particulartarget. The task of screening is to select from this large set the fewpotential lead compounds displaying activity in binding or in functionalassays. The principal drug discovery strategy widely applied to naturalcompound libraries in the pharmaceutical industry is to selectindividual compounds from the library at random and subject them to aseries of tests. Systematic screening procedures are thus required toimplement the rapid screening and scoring of an entire library ofsynthetic compounds, in practice often containing on the order of 10^ 7items.

[0179] In current practice, compounds are first cleaved and eluted fromtheir solid supports and are stored in microtiter plates. Further samplehandling in the course of screening relies primarily on roboticpipetting and transfer between different containers, typically wells inmicrotiter plates. While robotic workstations represent a step in thedirection of automating the process, they rely on the traditional formatof microtiter plates containing 8×12 wells and sample handling bypipetting and thus represent merely an incremental operationalimprovement. A significant additional consideration is the need toconserve reagent and sample by reducing the spatial scale of theanalytical procedures.

[0180] The present invention provides a set of operations to realizeintegrated sample handling and screening procedures for bead-basedcompound libraries in a planar format. This will significantly reducetime and cost due to reagent and sample volumes. The principal advantageof the methods of the present invention is that they provide a large setof fundamental operations to manipulate sets of beads in a planarformat, permitting the handling of beads between stations in amulti-step analytical procedure.

[0181] In particular, as previously described herein, the methods of thepresent invention facilitate the implementation of the followingpertinent procedures: transfer of samples from microtiter plates to aplanar electrochemical cell; formation of heterogeneous panels of targetsites adjacent to the substrate surface; solid phase binding assays; andisolation of specific beads from an array. In addition, the fundamentaloperations of the present invention provide the means to concatenatethese procedures on the surface of a planar electrode.

[0182] As described herein for hybridization assays, several variantsare possible. That is, binding assays may be performed by allowingprotein targets such as enzymes to bind to compounds on the surface of abead, either in suspension or arranged in a planar array. The commonpractice of combinatorial chemistry based on large porous carrier beadsaccommodates the concurrent handling of smaller beads to whose outersurface compounds are anchored via inert chemical spacers. Such smallbeads (up to 10 microns in diameter) are readily manipulated by themethods of the present invention. Large beads are used as labeledcompound storage containers.

[0183] Alternatively, binding between target and a radioactively orotherwise labeled probe may occur in solution, within microtiter platewells, if compounds have already been cleaved from their synthesissupport. In that case, probe-target complexes may be captured bycomplexation to encoded beads in each well, for example via thesecondary antibody method of coupling the protein target to abead-anchored antibody. Bead-captured probe-target complexes are thentransferred to the planar cell for proximity analysis and furtherprocessing as illustrated in FIG. 10. As shown in FIG. 10, probe-targetcomplexes 102 are allowed to form in solution. Antibody coated beads 104are added to the solution, resulting in a bead anchored complex 106. Thebead anchored complexes 106 are deposited onto electrode 108 from wells110, and a planar array of bead anchored complexes is formed. Whenfluorescent probes 114 are used, these impart fluorescence to the beadanchored complex, facilitating detection.

[0184] The methods and apparatus of the present invention are wellsuited to the task of identifying a small number of positive events in alarge set. The imaging of an entire array of probe-target complexes isfurther enhanced by proximity to an area detector, and by bead lensingaction. The isolation of a small number of positive scores from thearray is readily achieved, for example by applying optical tweezers, asdescribed herein. The large remainder of the array may then bediscarded. This in turn considerably reduces the complexity of applyingmore stringent tests, such as the determination of binding constants,because these may be restricted to the few retained beads. These testsmay be directly applied, without the need for additional sample transferto new containers, to the samples surviving the first screening pass.

EXAMPLE IX

[0185] Hybridization Assays in Planar Array Format

[0186] The present invention can be used to implement solid phasehybridization assays in a planar array format in a configuration relatedto that of a protein binding assay in which target molecules arechemically attached to colloidal beads. The methods of the presentinvention facilitate the formation of a planar array of different targetoligonucleotides for presentation to a mixture of strands in solution.Alternatively, the array may be formed subsequent to hybridization insolution to facilitate detection and analysis of the spatialdistribution of fluorescence or radioactivity in the array.

[0187] Considerable research and development is presently being investedin an effort to develop miniaturized instrumentation for DNA sampleextraction and preparation including amplification, transcription,labeling and fragmentation, with subsequent analysis based onhybridization assays as well as electrophoretic separation.Hybridization assays in planar array format are being developed as adiagnostic tool for the rapid detection of specific single base pairmutations in a known segment of DNA, and for the determination ofexpression levels of cellular genes via analysis of the levels ofcorresponding mRNAs or cDNAs. Hybridization of two complementary singlestrands of DNA involves molecular recognition and subsequent hydrogenbond formation between corresponding nucleobases in the two opposingstrands according to the rules A-T and G-C; here A, T, G and Crespectively represent the four nucleobases Adenine, Thymine, Guanosineand Cytosine found in DNA; in RNA, Thymine is replaced by Uracil. Theformation of double-strand, or duplex, DNA requires the pairing of twohighly negatively charged strands of DNA, and the ionic strength of thebuffer, along with temperature, plays a decisive role.

[0188] As previously discussed herein, two principal methods to prepareheterogeneous arrays of target strands on the surface of a planarsubstrate are micro-dispensing (“printing”) and in-situ, spatiallyencoded synthesis of oligonucleotides representing all possible sequencepermutations for a given total length of strand. In this context,hybridization must necessarily occur in close proximity to a planarsubstrate surface and this condition requires care if complications fromsteric hindrance and from non-specific binding of strands to thesubstrate are to be avoided. Non-specific adsorption can be a seriousproblem, especially in the presence of DC electric fields employed incurrent commercial designs that rely on electrophoretic deposition toaccelerate the kinetics of hybridization on the surface. In addition,there are the technical difficulties, previously discussed herein,resulting from steric hindrance and from collective effects reflectingthe crowding of probe strands near the surface.

[0189] In the context of DNA analysis, colloidal (magnetic) beads arecommonly used. For example, they are employed to capture DNA in a widelyused screening procedure to select cDNAs from clone libraries.Specifically, cDNAs are allowed to hybridize to sequences within longgenomic DNA that is subsequently anchored to magnetic beads to extractthe hybridized cDNA from the mixture.

[0190] The present invention facilitates the formation of planar arraysof oligonucleotide-decorated colloidal beads, either prior to orsubsequent to hybridization of a fluorescence probe strand to thebead-anchored target strand or subsequent to hybridization in freesolution and bead capture of the end-functionalized target strand. Incontrast to prior art methods, the present invention does not requirehybridization to occur in the vicinity of planar substrate surface,although this is an option if bead-anchored probe strands are to bedelivered to substrate-anchored target strands.

[0191] The ability to perform hybridization either in solution, on thesurface of individual beads, or at the substrate surface provides anunprecedented degree of flexibility. In addition, the advantages of beadarrays, as described herein, make it feasible to select and isolateindividual beads, or groups of beads, from a larger array on the basisof the score in a hybridization assay. This isolation facilitates theimplementation of subsequent assays on the strands of interest. The factthat beads remain mobile also means that beads of interest may becollected in designated holding areas for micro-sequencing, or may bemoved to an area of substrate designated for PCR amplification.

[0192] The methods of the present invention may be used to implement ahybridization assay in a planar array format in one of two principalvariations. All involve the presence of the entire repertoire of beadsin the planar array or panel formed adjacent to the electrode surfacefor parallel read-out. As with heterogeneous panels in general, thearrangement of beads within the array is either random (with respect tochemical identity), and the identity of beads scoring high in thebinding assay must be determined subsequently, or it is spatiallyencoded by invoking the “Layout-Preserving Transfer” method of sampleloading described herein.

[0193] The former variant is readily implemented and accommodates arrayformation either prior to or subsequent to performing the binding assay.For example, binding may be performed in suspension before beads areassembled into the array. As with the aforementioned cDNA selectionprocedure, the method of the present invention also accommodates the useof beads as capture elements for end-functionalized target DNA, forexample, via biotin-streptavidin complexation. In this latter case,beads serve as a delivery vehicle to collect all probe-target complexesto the electrode surface where they are assembled into an array for easeof analysis. In particular, proximity CCD detection of beads onelectrodes will benefit from the lensing action of the beads in thearray. This version of the assay is preferably used if only a smallnumber of positive scores are expected.

[0194] Hybridization to a pre-formed bead array can take advantage of avariant of the assay which preserves spatial encoding. An array of beadclusters is formed by the “Layout-Preserving Transfer” method previouslydescribed herein, and exposed to a mixture of cDNAs. The resultingspatial distribution of fluorescence intensity or radioactivity reflectsthe relative abundance of cDNAs in the mixture. This procedure relies onthe detection of a characteristic fluorescence or other signal from theprobe-target complex on the surface of a single bead. Given the factthat the array is readily held stationary by the methods of the presentinvention, image acquisition may be extended to attain robustsignal-to-noise for detection of low level signals. For example, asignal generated by a bead of 10 micron diameter with at most 10^ 8probe-target complexes on the surface of the bead may be detected. Beadlensing action also aids in detection.

[0195] As with the implementation of drug screening, the functionalelements of the present invention may be combined to perform multiplepreparative and analytical procedures on DNA.

EXAMPLE X

[0196] Alignment and Stretching of DNA in Electric Field-Induced Flow

[0197] The present invention can be used to position high-molecularweight DNA in its coiled configuration by invoking the fundamentaloperations as they apply to other colloidal particles. However, inaddition, the electrokinetic flow induced by an electric field at apatterned electrode surface may be employed to stretch out the DNA intoa linear configuration in the direction of the flow.

[0198] Procedures have been recently introduced which rely on opticalimaging to construct a map of cleavage sites for restriction enzymesalong the contour of an elongated DNA molecule. This is generally knownas a “restriction map”. These procedures, which facilitate the study ofthe interaction of these and other proteins with DNA and may also leadto the development of techniques of DNA sequencing, depend on theability to stretch and align DNA on a planar substrate.

[0199] For individual DNA molecules, this has been previously achievedby subjecting the molecule to elongational forces such as those exertedby fluid flow, magnetic fields acting on DNA-anchored magnetic beads orcapillary forces. For example, DNA “combs” have been produced by simplyplacing DNA molecules into an evaporating droplet of electrolyte. Ifprovisions are made to promote the chemical attachment of one end of themolecule to the surface, the DNA chain is stretched out as the recedingline of contact between the shrinking droplet and the surface passesover the tethered molecules. This leaves behind dry DNA molecules thatare attached in random positions within the substrate area initiallycovered by the droplet, stretched out to varying degrees and generallyaligned in a pattern of radial symmetry reflecting the droplet shape.Linear “brushes”, composed of a set of DNA molecules chemically tetheredby one end to a common line of anchoring points, have also beenpreviously made by aligning and stretching DNA molecules bydielectrophoresis in AC electric fields applied between two metalelectrodes previously evaporated onto the substrate.

[0200] The present invention invokes electrokinetic flow adjacent to anelectrode patterned by UV-mediated regrowth of oxide to provide a novelapproach to the placement of DNA molecules in a predeterminedarrangement on a planar electrode surface, and to the stretching of themolecules from their native coil configuration into a stretched, linearconfiguration that is aligned in a pre-determined direction. Thisprocess is shown in FIG. 11 and is accomplished by creating controlledgradients in the flow vicinity across the dimension of the DNA coil. Thevelocity gradient causes different portions of the coil to move atdifferent velocities thereby stretching out the coil. By maintaining astagnation point at zero velocity, the stretched coil will be fixed inposition. This method has several advantages over the prior artapproaches. First, DNA molecules in their coiled state are subjected tolight control to form arrays of desired shape in any position on thesurface. This is possible because large DNA from cosmids or YACs formscoils with a radius in the range of one micron, and thus acts in amanner analogous to colloidal beads. A set of DNA molecules may thus besteered into a desired initial arrangement. Second, UV-patterningensures that the elongational force created by the electrokinetic flowis directed in a predetermined direction. The presence of metalelectrodes in contact with the sample, a disadvantage of thedielectrophoretic prior art method, is avoided by eliminating thissource of contamination that is difficult to control especially in thepresence of an electric field. On patterned Si/SiOx electrodes, flowvelocities in the range of several microns/second have been generated,as required for the elongation of single DNA molecules in flow. Thus,gradients in the flow field determines both the fractional elongationand the orientation of the emerging linear configuration. Third, thepresent invention facilitates direct, real-time control of the velocityof the electric field-induced flow, and this in turn conveys explicitcontrol over the fractional elongation.

[0201] This invention is also for a method and apparatus to direct thelateral motion and induce the assembly into planar arrays of cells onsemiconductor surfaces in response to temporally and spatially varyingelectric fields and to projected patterns of illumination.

[0202] The response of different types of cells to variations in thefrequency and voltage of the applied electric field and to specificpatterns and changing intensity of illumination enables the on-cueassembly of multiple arrays of cells in any desired position on thesubstrate, and further enables the repositioning, disassembly andre-assembly and more generally the reconfiguration and segmentation ofsuch arrays. Time-constant and time-varying illumination patterns aregenerated by an optically programmable illumination pattern generatormay be used and can be based on a graphical representation of thepattern created on a personal computer.

[0203] The novel methodology of image cytometry introduced in thepresent invention relates to the general fields of molecular andcellular biology and has applications in the fields of diagnostics,genetic analysis, cell biology and drug discovery. The image cytometryfacilitates a multiplicity of cell-based biological and biochemicalprocedures based on the quantitative analysis of images recorded fromarray of cells. As with Random Encoded Array Detection (READ) orProgrammable Array Reconfiguration and Segmentation (PARSE), theseassays are performed in a highly parallel format in a small volume ofsample on the surface of a semiconductor wafer.

[0204] The novel method of image cytometry according to the presentinvention may be used in a number of different applications, including:

[0205] cell-typing analysis based on the decoration of cell-surfacemarkers with tagged probes such as fluorescence labeled antibodies orantibodies that are coupled to flourescent beads. Specific applicationsinclude bacterial strain typing; HLA typing (transplantationdonor-recipient matching); immunophenotyping;

[0206] diagnostic assays based on the identification of cell types andquantitative evaluation of relative counts of different cell types in agiven sample, for example, the determination of the fraction of CD-4cells in a population to assess levels of HIV infection;

[0207] on-chip fractionation of a mixture of cells into sub-populationsbased on:

[0208] selective affinity of cell phenotypes using antibodies or lectin(Nordon et al, “An Experimental Model of Affinity Cell Separation”,Cytometry 16, 25-33 (1994), the contents of which are incorporatedherein by reference; see also: “panning” for clones expressing specificantibodies). Using beads displaying such antibodies or lectins,cross-linking between cells may be induced to form clusters that arereadily distinguished and separated from individual cells;

[0209] differential mobility in response to time-varying or totime-constant but spatially varying (inhomogeneous) electric fields orto gradients in electrochemical environment created by LEAPS near anelectrolyte-insulator-semiconductor (EIS) interface;

[0210] combinations with controlled flow configurations such as those invarious realizations of field flow fractionation (for example, see:Bigelow et al, “Separation of B and T lymphocytes by a hybrid field-flowfractionation/adhesion chromatography technique”, J. ImmunologicalMethods 117, 289-293 (1989); Markx et al, “Dielectrophoreticcharacterization and separation of microorganisms”, Microbiology 140,585-591 (1994); Huang et al, “Introducing Dielectrophoresis as a NewForce Field for Field Flow Fractionation”, Biophysical J. 73, 1118-1129(1997); the contents of all of which are incorporated herein byreference);

[0211] cell-based functional assays monitoring the levels of expressedcell-surface markers or the activation or regulation of pathways, suchas signal transduction pathways and metabolic pathways. The expressionof the surface markers may be detected by affinity binding between themarkers and fluorescence or enzyme labeled probes. The endpoints of thepathways may be analyzed through intracellular fluorescent signalgenerated by enzymes such as luciferin (PE Corporation/Tropix) orβ-lactamase (Aurora);

[0212] cell secretion-based analysis that could be used in drug andligand screening;

[0213] cell-cell interactions revealed by adhesion markers on cellsurfaces;

[0214] cytotoxicity assays based on the labeling of indicators such asphosphatidylserine which appears on the outer membrane of cells in theearly stages of apoptosis; and

[0215] chemotaxis analysis based on the tracking of cells positioned incontrolled configurations in known spatial relation to gradients in theconcentration of chemotactic agents.

[0216] The image cytometry according to the present invention permitsthe efficient realization of a wide variety of cell-based functional aswell as diagnostic assays based on the analysis of multi-colorfluorescence or other optical markers imaged on the surfaces ofindividual cells. In addition, the methodology also permits theseparation and fractionation of multiple cell types in a mixture basedon differential response to electro-optic stimuli as disclosed herein.Imaging and profiling applications may invoke a variation of the READformat while fractionation and sorting into multiple sub-populations mayinvoke the PARSE format.

[0217] The integration of this novel methodology of cellular analysiswith sample preparation (electric-field induced lysis of cells) alongwith the technology of optically programmable bead arrays as disclosedherein creates a novel platform that combines an “on-chip” format ofquantitative molecular interaction analysis and cellular analysis withmicrofluidics and optical imaging into a novel, multi-purpose assayplatform.

[0218] Imaging of cells—In its simple form, image cytometry is practicedin the doctor's office when blood or other liquid samples are spottedonto segmented glass slides and populations of cells are “manually”counted. These and related simple implementations of cellular analysisinvoke the simple spotting of cells onto solid supports where cellssettle under gravity, typically in random positions at low lateraldensity. However, even in this simple format, certain advantages of theapproach have been recognized (Galbraith et al, “Imaging Cytometry byMultiparameter Fluorescence”, Cytometry 12, 579-596 (1991), the contentsof which are incorporated herein by reference): the power of imageanalysis can be brought to bear to facilitate the analysis ofmulti-color fluorescence images. A major drawback is the very low“throughput” of this approach, given the low density of cells which mustbe maintained to avoid the risk of forming three-dimensional clusters inwhich the view of some of the cells would be obstructed. A furtherdrawback is that methods of cellular analysis invoking differentialmobility of cells in response to external stimuli are not available.

[0219] Flow cytometry—Flow cytometry and its application tofluorescence-activated cell sorting rely on a serial mode of analysisthat requires the capture of individual cells into droplets which arepassed, one at a time, through a small observation window. During ashort time, e.g., 1-10 μs, all measurements on a given cell must becompleted. For example, the simultaneous analysis of a small number ofcell surface markers represents the state of the art in flow cytometry(Beavis and Pennline, “Detection of Cell-Surface Antigens UsingAntibody-conjugated Fluorospheres”, BioTechniques 21, 498-503 (1996),the contents of which are incorporated herein by reference). Flowcytometric analysis represents just the last step in an assay: theinstrumentation does not lend itself to the integration of liquidhandling and automation of bioanalytical assays. This is in significantcontrast to the present invention which integrates the opticalmanipulation of bead arrays and cellular assembly technology withmicrofluidics and optical imaging into a miniaturized platform forquantitative molecular and cellular analysis.

[0220] Recent developments of smaller and more specialized instrumentsnotwithstanding, the intrinsic complexity of flow cytometry requiresspecialized and expensive instrumentation including multiple lasersources of high power, matching detectors and fast electronics to handlethe acquisition and processing of signals during the short availableinterrogation interval during which each cell traverses the detectionwindow. Sorting requires additional steps and equipment. Even after manyyears of commercial development, the operation of flow cytometersrequires trained personnel to handle calibration and ensure reliableperformance. The cost of commercial instruments, for example, thoseoffered by Becton-Dickinson, Beckman Coulter or Cytomation isprohibitive for all but major medical centers or research institutions.

[0221] Sorting and separation of cells by affinity and/or flow—Severalrealizations have been described for the fractionation of cells byselective affinity for, and hence differential retention by a nearbysolid phase or by differential mobility in superimposed force fieldssuch as a fluid flow with superimposed transverse electric field (fieldflow fractionation or FFF) or by combinations thereof. Differentialdielectrophoretic levitation of cells above a set of interdigitatedelectrodes and fractionation in a transverse flow represents a recentaddition to this set of techniques which offers the potential fordifferential blood cell analysis. As with other FFF techniques, thelatter method relies on downstream detection of individual cells andthus resembles flow cytometry with respect to the implied serialprocessing to establish the identity of sub-populations. A furtherdisadvantage is the requirement for an elongated channel to permit thespatial separation between sub-populations.

Cytometry According to the Present Invention

[0222] Cytometry according to the present invention subsumes the entireset of capabilities for cellular analysis illustrated above into acommon platform which is miniaturized and integrated with microfluidicsand optical imaging and compatible with quantitative bear array formatof molecular interaction analysis whose capabilities may be used in“multiplexed” fluorescence immunoassays and DNA hybridization assays.

[0223] The specific advantages of the cytometry according to the presentinvention include:

[0224] high throughput resulting from the high densities of the cellularassemblies (for example, 10^ 4-10^ 6/mm^ 2) and the parallel mode ofsignal acquisition afforded by imaging;

[0225] integration of sample preparation, fluid handling and assayreadout, for example, integration with on-chip assay formats;

[0226] parallel processing based on imaging of multiple colors andmorphological parameters;

[0227] light-directed transport and assembly of cells;

[0228] light-guided separation and fractionation of cells intosub-populations based on optically programmable illumination patternsand frequency dispersion in the dielectrophoretic response; and

[0229] significant reduction in expense of instrumentation.

[0230] The method and apparatus of the present invention enables theparallel processing of large numbers of cells assembled into denseplanar assemblies; typically, 10^ 4 cells of 10 μm diameter will beaccommodated in an area as small as 1 mm^ 2. Smaller bacterial cellssuch as E.coli can be accommodated in even higher density. Insignificant contrast to flow cytometry, signals are collectedsimultaneously from a large number of cells by way of imaging, and therate of acquiring signal is very high even when cells are heldstationary.

[0231] The present invention presents a very significant advantage,especially when multiple colors are to be discriminated and are to bequantitatively determined by analysis of multi-color images ofindividual cell surfaces. Thus, the cytometry according to the presentinvention will readily accommodate multiple cell surface markers andadditional morphological indicators for individual cells (size, shape)or mutually interacting clusters of cells. This is generally possibleeven without specialized equipment. In fluorescence immunoassays usingthe READ bead array format, the capability has been demonstrated usingan inexpensive consumer-grade video CCD camera, of detecting signalsfrom individual beads displaying only 1000 labeled antibody molecules.Higher levels of sensitivity are possible for detection of markers oncell surfaces because fluorescent beads may be used as the label todecorate the cells.

[0232] A. Cells form Arrays in Response to AC Electric Fields

[0233] Referring now to FIG. 12, therein is illustrated E. coli cells(grown to log phase and suspended in 280 mM mannitol solution at thedensity of 10⁸/ml) which form arrays under electric field. On the leftside of the field, the cells are assembled at very high density. Somesinglet cells at the right side are moving to the left to join thearray. (Bright field image. 25× obj, NA 0.45. Electric field: 5V, 200Hz).

[0234] Referring now to FIG. 13, therein is illustrated the same effectas in FIG. 12, with S. cerevisiae cells (baker's yeast). (Bright fieldimage. 25× obj, NA 0.45; electric field: 5V, 400 Hz).

[0235] B. Cells Assemble in Accordance with Projected IlluminationPatterns.

[0236] Referring now to FIG. 14, therein is illustrated E. coli formingdiamond shapes that are projected to the surface by an LCD panelcontrolled by appropriate graphics software; (20× objective, NA 0.28;Electric field: 10V, 7 kHz).

[0237] Referring now to FIG. 15, therein is illustrated S. cerevisiaeresponding to two circular illumination patterns;(20× objective, NA0.28; Electric field: 15 V, 15 kHz).

[0238] C. Light-directed Cell Transport and Reconfiguration of CellularAssemblies

[0239] By enabling the controlled transport of cells and cellularassemblies in accordance with the PARSE format, the cytometry accordingto the present invention provides an entire additional level of cellularanalysis which has the inherent capability to fractionate cellpopulations “in-situ”.

[0240] The principal approach according to the present invention is togenerate gradients in electrochemial properties of the interface towhich sub-populations of cells exhibit a differential response. Suchgradients may be produced by invoking patterns of illumination ingeometry, as disclosed herein. Examples in the figures illustrate thatcells respond to these patterns in a variety of ways.

[0241] For example, cells may be collected into illuminated regions(FIG. 16; S. cerevisiae responding to the illumination. Arrow indicatethe direction cells are moving in. (20× objective, NA 0.28; Electricfield: 15 V, 15 kHz)) or expelled from illuminated regions (FIG. 17; E.Coli cells being repelled from the illumination circle.(20× objective,NA 0.28; Electric field: 10 V, 7 kHz; low substrate doping level (1 Ohmcm-10 Ohm cm)); “channeled” by reconfiguring illuminated regions or“dragged” and “dropped” by displacing illuminated regions (FIG. 18; E.coli cells following the expansion of the rectangular illuminated shape(20× objective, NA 0.28; Electric field: 15 V, 8 kHz)).

[0242] Cells also may be “trapped” in well defined regions in responseto light-induced spatial gradients in the electrical properties of theEIS structure (FIG. 19; E. coli cells being trapped by the light stripeat the left and being prevented from moving to the circular region onthe right. (20× objective, NA 0.28; Electric field: 15 V, 8 kHz)). Thisset of fundamental operations permits the creation of spatially varyingelectrochemical potentials and corresponding lateral, highlyinhomogeneous electric fields. In addition, the cytometry according tothe present invention permits the programmable lateral displacement andreconfiguration of illumination patterns so that the location and thestrength of the spatial inhomogeneities in electric fields can beadjusted at will.

[0243] In addition, the cytometry according to the present inventionalso provides frequency-dependent forces to which different constituentswithin mixtures of cells will generally exhibit differential responses.An example of segregation between different types of cells is shown inFIG. 20 As shown in FIG. 20, when an illuminated region containing bothtypes of cells is suddenly expanded or “dragged ”, E. coli cells (left)respond faster than the larger S. cerevisiae cells (right), resulting insegregation. (Bright field image. 25× obj, NA 0.45 Electric field: 5V,400 Hz).

[0244] Specifically, on the basis of the known dielectrophoreticproperties of various cells, fractionation will be possible on the basisof characteristic frequency dispersion. For example, the characteristictransition of cells from positive to negative dielectrophoresis (asevidenced in electrorotation; see e.g.: Huang et al, op. cit.) will bethe basis for the fractionation of cell mixtures in light-inducedgradients in electrochemical EIS potential (and the resultinginhomogeneous fields): different cells will be expelled from confinedregions on the substrate at characteristic frequencies and lightintensities.

[0245] D. Non-viable Cells Behave Differently than the Viable Cells.

[0246] The present inventors have observed that heat treated S.cerevisiae cells stop responding to the electric field and light afterthe first ten to twenty seconds and adhere to the substrate.

EXAMPLE

[0247] Immunophenotyping—In a manner analogous to READ, a cellular arrayformat will provide a sensitive and highly parallel mode of monitoringthe presence of characteristic cell surface markers as illustrated inFIG. 21. For example, groups of lymphocyte cells can be put into arrayformat on the substrate. Antibodies for the cell surface markers (CD3,CD4, CD8, CD56, etc.) are coupled to small microspheres that aredifferentially coded. The mixture of beads with different antibodies aredirected to the cells arrays using the optical arrangement principles ofthe present invention. Antibodies on the bead surface will bind to thecell surface markers, thus, the correspondent beads will link to thecells. The number of surface markers which can be detectedsimultaneously depends on chemical coding capacity of the bead. Hundredsof chemical codings may possibly be used. Signal integration capabilityaccording to the present invention enables more accurate andquantitative analysis of the cells.

EXAMPLE

[0248] Multiplexed Affinity Detection—A novel format enabling thesimultaneous affinity identification of multiple types of cells is shownin FIG. 22. A random encoded bead array of antibodies will provide anovel, highly diverse set of anchoring points for lymphocytes displayingcognate cell surface antigens. Under conditions of controlledstringency, flow-induced shear will remove cells that are notspecifically bound while leaving “positives” attached to individualbeads. Focusing on this downstream step takes advantage of the intrinsic“concentration” of sample on the cell surface and the ability tocapture, with a single antibody probe, multiple pathogens sharing commonprocessed antigenic determinants.

EXAMPLE

[0249] Light-guided Sorting and Isolation—Combination of AC electricfields with light-controlled modulation of surface-electrochemicalproperties is particularly powerful. The illuminated areas mimics thebehavior of patterned electrodes and can be repositioned in real time toisolate designated cells. Cells can be sorted and isolated due to theirnatural properties or due to the property change caused by specificbinding to probe labeled beads. The model is illustrated in FIG. 23.Cells can be recovered after the isolation and further culturing oranalysis may be performed. Specific lymphocyte cells and cancer cellsmay be of particular interest for such cell sorting and analysis.

EXAMPLE

[0250] Cell-based Functional Assays—Cell-based functional assays may beperformed in a highly efficient manner by assembling cellular arrays andimaging reaction of the cells as shown in FIG. 24. The reaction may bethe direct change of extracellular markers, or the endpoints of somemetabolic pathways. The endpoints may be intracellular fluorescencesignal generated by enzymes such as β-lactamase whose substrate canpenetrate the cells, thus, the signal may be directly quantitativelydetected by imaging (Zlokarnik et al., “Quantitation of Transcriptionand Clonal Selection of Single Living Cells with β-lactamase asReporter”, Science 279, 84-88 (1998); see also: Bronstein et al,“Chemiluminescent and Bioluminescent Reporter Gene Assays”, Anal.Biochemistry 219, 169-181 (1994); the contents of all of which areincorporated herein by reference). If combined with microfludic control,such cell-based functional assays have tremendous applications in drugscreening and ligand analysis. Cells may be placed at the same locationas the chemical carrier beads. Under temporal and spacial controlledrelease of the compounds or ligands, response of the cells may berecorded. After image analysis, compounds or ligands that generate thedesired cell response may be easily identified. Because the cells may bedirectly imaged, cell lysis and the following assays will generally notbe necessary as part of the traditional cell-based analysis.Furthermore, the controlled assembly of the cells and chemical carryingbeads, according to the present invention, may eliminate the use ofmicrotiter plates in the cell-based screening.

EXAMPLE

[0251] Integrated Format Profiling of Secreted Cellular Markers—Controlof cell assembly and microfluidic pathway enables direct in situscreening of secreted cellular markers. This model is illustrated inFIG. 25. Cellular arrays are assembled in one compartment of atwo-compartment liquid reactor and are exposed to candidate drugs; theresponse is measured by monitoring a panel of secreted markers such ascytokines. Cell secretion are collected and introduced into a secondcompartment where they are profiled based on the response of apre-deposited bead array using the READ format. Such a two-compartmentreactor may be operated in parallel for multiple drug screening orkinetic analysis.

EXAMPLE

[0252] Cell-to-Cell Interaction—Cell-to-cell in vivo interaction wasseldom studied directly due to the lack of control in cell manipulation.The present invention provides a powerful tool in this area. Asillustrated in FIG. 26, cells may be temporally and spatially controlledto favor the binding between the cells. Alternatively, cells may bedirected to preassembled cell arrays for cell binding analysis. It mayalso be possible to study the interaction between cells such as T cells,B cells and polymorphonulear leukocytes, in the activation of immuneresponses.

EXAMPLE

[0253] Chemotaxis Analysis—Cells can be positioned in a controlledconfiguration to test their chemotaxis properties. This model isillustrated in FIG. 27. For example, D. discoideum cells may be testedunder cAMP or specific polypeptide gradient. (Parent & Devreotes, “Acell's sense of direction”, Science 284, 765-770 (1999); the contents ofwhich are incorporated herein by reference). Response of the receptors,G proteins, actin or actin-binding proteins may be detected throughfluorescence signal generated by their correspondent fusion protein withGFP (Green Fluorescent Protein). Movement of the cells may also berecorded. More importantly, the force of the movement may be measured bycomparing to the energy used in the light control of the cells.

[0254] The present invention may be used for cell identification anddetection, for example, in immunophenotyping and affinity celldetection. In this application, affinity probes are coupled to beads ona solid surface. The beads are chemically and spatially coded. The cellsare transported to the desired position using light control. Cells withthe surface markers will bind at the correspondent positions. Theadvantages of this approach include a simpler and less expensive system,a higher degree of throughput for parallel immunophenotyping, themanipulation of a large number of cells, thus the detection of rareevents, and better integration between the reaction and the detectionsteps, thus allowing the entire process to be performed on-chip.

[0255] The present invention may also be used in cell sorting,fractionation and isolation. In this case, cells can be separatedaccording to their intrinsic properties, such as their dielectrophoreticmobility, viability, size, etc. Cells can also be sorted by theirassociation with antibody coupled beads. The advantages of this approachinclude multiple cell sorting, simpler substrate fabrication, andflexible and programmable control.

[0256] The present invention may also be used for screening, forexample, cell mediated drug and ligand screening, or cell secretionmediated screening. In this approach, cells may be transported andassembled a the desired position. Chemical compounds or ligands arereleased at the corresponding position. The response of the cells maythen be observed through their reporter genes. The advantages of thisapproach include higher throughput, a simpler system without thecomplication of automated pipetting machines, and the possiblecombination with microscopy for simpler and more direct assays.

[0257] The present invention may also be used for cell to cellinteraction, for example, in suspension or on surface interaction. Inthis approach, cells may link with each other through their surfaceinteraction. Movement of the cells may then be guided by light. Theadvantages of this approach include the ability to study the interactionbetween cells.

[0258] While the invention has been particularly shown and describedwith reference to a preferred embodiment thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the spirit and scope of theinvention.

What is claimed is:
 1. An assembly of cells comprising: a substrate; anda dynamically formed planar assembly of cells in a designated area onthe substrate.
 2. The assembly of cells accordingly to claim 1, whereinsaid assembly is formed by a method comprising the following steps: (a)providing a first electrode positioned in a first plane and a secondelectrode positioned in a second plane different from the first plane;(b) providing a plurality of cells suspended in an electrolyte solution,the cells and the electrolyte solution being located between the firstand the second electrode, wherein the second electrode comprises either(i) a light-sensitive electrode, such that when an AC electric field isgenerated at an interface between the second electrode and theelectrolyte solution and the second electrode is illuminated with apredetermined light pattern, a planar assembly of cells forms in adesignated area of the second electrode, said designated area beingdefined by the pattern of illumination or (ii) a electrode having asurface or an interior, the surface or interior having been modified toproduce spatial modulation in properties of the second electrode thataffects the local distribution of an electric field generated at aninterface between the second electrode and the electrolyte solution,such that when an AC electric field is generated at the interface, aplanar assembly of cells forms in a designated area of the secondelectrode, said designated area being defined by the spatial modulationin the properties of the second electrode; and (c) forming a planarassembly of cells in a designated area of the second electrode byapplying an AC electric field at the interface between the secondelectrode and the electrolyte solution and, if the second electrodecomprises a light-sensitive electrode, illuminating the second electrodewith a predetermined light pattern.
 3. The method of claim 2, whereinthe first and the second electrode each comprises a planar electrodethat are parallel to each other and separated by a gap, with theelectrolyte solution and the cells being located in said gap, whereinthe electric field is generated by applying an AC voltage between theelectrodes.
 4. The method of claim 3, wherein the first electrodecomprises an optically transparent electrode that allows optical imagingof the cell assembly.
 5. The method of claim 2, wherein the first andthe second electrode are contained in an assay cartridge, said cartridgehaving a fluidic connection that permits introduction of a fluid intothe cartridge and exit of the fluid from the cartridge.
 6. The method ofclaim 2, wherein the second electrode comprises a silicon electrode. 7.The method of claim 2, wherein the second electrode comprises a siliconelectrode coated with a dielectric layer.
 8. The method of claim 2,wherein the second electrode comprises a light-sensitive electrode, saidelectrode having been patterned by spatially modulated oxide growth,surface chemical patterning or surface profiling, such that the locationof the cellular assembly is controlled by said patterning in addition tothe illumination.
 9. The method of claim 2, wherein the second electrodecomprises an electrode patterned by spatially modulated oxide growth,surface chemical patterning or surface profiling, such that the locationof the cellular assembly is controlled by said patterning.
 10. Themethod of claim 2, wherein the designated area corresponds to an area oflow interfacial impedance.
 11. The method of claim 2, wherein the cellsin the assembly are immobilized by chemical or physical means.
 12. Themethod of claim 2, wherein the cellular assembly has a density of atleast about 10⁴ cells/mm².
 13. A method of detecting a bindinginteraction between a cell and an analyte, the method comprising:providing a dynamically formed planar assembly of cells according toclaim 1; contacting the cells with an analyte to allow binding of theanalyte to the cells; and detecting said binding.
 14. A method ofdetecting a binding interaction between a cell and an analyte, saidmethod involving an assembly of cells according to claim 2 andcomprising the following steps: (a) providing a first electrodepositioned in a first plane and a second electrode positioned in asecond plane different from the first plane; (b) providing a pluralityof cells suspended in an electrolyte solution, the cells and theelectrolyte solution being located between the first and the secondelectrode, wherein the second electrode comprises either (i) alight-sensitive electrode, such that when an AC electric field isgenerated at an interface between the second electrode and theelectrolyte solution and the second electrode is illuminated with apredetermined light pattern, a planar assembly of cells forms in adesignated area of the second electrode, said designated area beingdefined by the pattern of illumination or (ii) a electrode having asurface or an interior, the surface or interior having been modified toproduce spatial modulation in properties of the second electrode thataffects the local distribution of an electric field generated at aninterface between the second electrode and the electrolyte solution,such that when an AC electric field is generated at the interface, aplanar assembly of cells forms in a designated area of the secondelectrode, said designated area being defined by the spatial modulationin the properties of the second electrode; (c) contacting the cells withan analyte to allow binding of said analyte to the cells; (d) forming aplanar assembly of cells in a designated area of the second electrode byapplying an AC electric field at the interface between the secondelectrode and the electrolyte solution and, if the second electrodecomprises a light-sensitive electrode, illuminating the second electrodewith a predetermined light pattern, wherein the planar assembly of thecells are formed either before or after the contacting of the cells withthe analyte; and (e) detecting the binding of the analyte to the cells.15. The method of claim 14, wherein the analyte is directed to aspecific cellular marker, and step (e) comprises detecting the presenceof the cellular marker on the surface of the cells.
 16. The method ofclaim 15, wherein the bioassay is directed to cell typing, with thepresence of the marker on the cell surface indicating the cell type. 17.The method of claim 14, wherein the analyte comprises a ligand directedto a specific cellular receptor and step (e) comprises detecting thepresence of the receptor on the surface of the cells.
 18. The method ofclaim 14, wherein the analyte comprises an antibody directed to aspecific cellular antigen and step (e) comprises detecting the presenceof the antigen on the surface of the cells.
 19. The method of claim 14,wherein the analyte is attached to a bead, said bead being associatedwith a chemically or physically distinguishable characteristic thatuniquely identifies the analyte attached thereto.
 20. The method ofclaim 19, wherein said bead is associated with a fluorescent tag thatuniquely identifies the analyte attached thereto.
 21. The method ofclaim 14, wherein a plurality of analytes is tested simultaneously forbinding with the cells, with each analyte being attached to a bead thatis distinguishable by chemical or physical characteristic, and thedetecting step comprising optically imaging the planar assembly of cellsand decoding the beads bound to the cells to determine the respectiveidentities of analytes bound to the cells.
 22. The method of claim 14,wherein the analyte comprises a ligand or an antibody attached to abead, said bead being associated with a chemically or physicallydistinguishable characteristic that uniquely identifies the analyteattached thereto.
 23. The method of claim 22, wherein step (d) resultsin formation of a mixed assembly comprising the cells and the beads in adesignated area on the second electrode, the proximity of the cells withthe ligand or the antibody attached to the bead allowing the bindinginteraction therebetween, and wherein the method further comprisesdisassembling said mixed assembly of the cells and the beads anddetecting the binding interaction by analyzing the formation of clusterscomposed of the cells and the beads, the binding indicating the presenceof absence of a cellular receptor or an antigen specific for the ligandor the antibody.
 24. The method of claim 22, further comprisingproviding a planar array of said beads in a designated area on thesecond electrode, wherein the cells are assembled in the same designatedarea as the bead array and the binding interaction is detected byselective adhesion of the cells to the beads in the array.
 25. A methodof detecting a cellular response to an analyte, the method comprisingproviding a dynamically formed planar assembly of cells according toclaim 1; contacting the cells with an analyte; and detecting a cellularresponse to the analyte.
 26. A method of detecting a cellular responseto an analyte, the method involving an assembly of cells according toclaim 2 and comprising the following steps: (a) providing a firstelectrode positioned in a first plane and a second electrode positionedin a second plane different from the first plane; (b) providing aplurality of cells suspended in an electrolyte solution, the cells andthe electrolyte solution being located between the first and the secondelectrode, wherein the second electrode comprises either (i) alight-sensitive electrode, such that when an AC electric field isgenerated at an interface between the second electrode and theelectrolyte solution and the second electrode is illuminated with apredetermined light pattern, a planar assembly of cells forms in adesignated area of the second electrode, said designated area beingdefined by the pattern of illumination or (ii) a electrode having asurface or an interior, the surface or interior having been modified toproduce spatial modulation in properties of the second electrode thataffects the local distribution of an electric field generated at aninterface between the second electrode and the electrolyte solution,such that when an AC electric field is generated at the interface, aplanar assembly of cells forms in a designated area of the secondelectrode, said designated area being defined by the spatial modulationin the properties of the second electrode; (c) contacting the cells withan analyte; (d) fonning a planar assembly of cells in a designated areaof the second electrode by applying an AC electric field at theinterface between the second electrode and the electrolyte solution and,if the second electrode comprises a light-sensitive electrode,illuminating the second electrode with a predetermined light pattern,wherein the planar assembly of the cells are formed either before orafter the contacting of the cells with the analyte; and (e) detecting acellular response to the analyte.
 27. The method of claim 26, whereinthe cellular response being detected comprises cellular secretion of oneor more biologically active substances, said secretion being induced bysaid analyte.
 28. The method of claim 27, wherein the biologicallyactive substance comprises a cytokine.
 29. A method of determining acellular response to an analyte, the method involving an assembly ofcells according to claim 2 and comprising the following steps: providinga planar array of beads on the first electrode, the beads displayingligands on their surfaces and each of said beads being associated with achemical or physical characteristic that uniquely identifies the ligandattached thereto, said ligands being attached to the beads in areleasable manner; forming a planar assembly of cells, with the gap areaseparating the beads from the assembled cells; and releasing the ligandsand monitoring a cellular response, the proximity of the cells to thebead array permitting determination of the identity of the ligandsinducing the cellular response.
 30. The method of claim 26, wherein theanalyte comprises a drug molecule or a ligand.
 31. The method of claim26, in which the cellular response being detected is an expression of aparticular gene, said expression being determined by detecting for thepresence of an intracellular reporter gene product.
 32. The method ofclaim 31, in which the expression of the intracellular reporter geneyields intracellular fluorescence.
 33. The method of claim 26, in whichthe cellular response being detected is selected from the groupconsisting of: morphological change of the cells, change in the cellmembrane permeability, and a change in the chemotaxis response bymonitoring the movement of cells.
 34. A method of determining aninteraction between different cell types, the method comprising thefollowing steps: providing a dynamically formed planar assembly of cellsaccording to claim 1, wherein the cells of the assembly comprises atleast two different cell types, with the proximity of one cell type toanother allowing an interaction between the different cell types tooccur; and detecting the cell-cell interaction.
 35. A method ofdetermining an interaction between different cell types, the methodinvolving the assembly of cells according to claim 2 and comprising thefollowing steps: (a) providing a first electrode positioned in a firstplane and a second electrode positioned in a second plane different fromthe first plane; (b) providing a plurality of cells comprising at leasttwo cell types suspended in an electrolyte solution, the cells and theelectrolyte solution being located between the first and the secondelectrode, wherein the second electrode comprises either (i) alight-sensitive electrode, such that when an AC electric field isgenerated at an interface between the second electrode and theelectrolyte solution and the second electrode is illuminated with apredetermined light pattern, a planar assembly of cells forms in adesignated area of the second electrode, said designated area beingdefined by the pattern of illumination or (ii) a electrode having asurface or an interior, the surface or interior having been modified toproduce spatial modulation in properties of the second electrode thataffects the local distribution of an electric field generated at aninterface between the second electrode and the electrolyte solution,such that when an AC electric field is generated at the interface, aplanar assembly of cells forms in a designated area of the secondelectrode, said designated area being defined by the spatial modulationin the properties of the second electrode; (c) forming a planar assemblyof cells in a designated area of the second electrode by applying an ACelectric field at the interface between the second electrode and theelectrolyte solution and, if the second electrode comprise alight-sensitive electrode, illuminating the second electrode with apredetermined light pattern, wherein the cells in the assembly comprisedifferent types of cells, with the proximity of one cell type to anotherallowing an interaction between the different cell types to occur; and(d) detecting the cell-cell interaction.
 36. The method of claim 35,wherein the interaction between the different cell types results in: amorphological change of at least one cell type; change in a cell surfacemarker of at least one cell type; change in a gene expression of atleast one cell type; a change in the membrane permeability of at leastone cell type; or adhesion of the cells to form a cluster.
 37. Themethod of claim 35, wherein step (d) comprises detecting the interactionbetween the cell types by ascertaining the formation of cell clusters.38. The method of claim 35, wherein the assembly of the mixed cell typesis prepared by the following steps: forming an assembly of a first celltype in a designated area on the second electrode; introducing cells ofa second type to the electrolyte solution and moving said cells to thedesignated area on the second electrode in which the first cell type islocated, thereby allowing the interaction between the two cell types tooccur.
 39. The method of claim 35, wherein the assembly of the mixedcell types is prepared by the following steps: forming an assembly of afirst cell type in a first designated area on the second electrode,forming an assembly of a second cell type in a second designated area onthe second electrode; and combining the assembly of the first and thesecond cell type to allow the interaction between the different celltypes to occur.